Microfluidic protein crystallography

ABSTRACT

The use of microfluidic structures enables high throughput screening of protein crystallization. In one embodiment, an integrated combinatoric mixing chip allows for precise metering of reagents to rapidly create a large number of potential crystallization conditions, with possible crystal formations observed on chip. In an alternative embodiment, the microfluidic structures may be utilized to explore phase space conditions of a particular protein crystallizing agent combination, thereby identifying promising conditions and allowing for subsequent focused attempts to obtain crystal growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 11/006,522 filed Dec. 6, 2004, now U.S. Pat. No. 7,459,022which claims priority of U.S. provisional patent application No.60/572,060 filed May 18, 2004. Also, U.S. patent application Ser. No.11/006,522 is a continuation-in-part of U.S. patent application Ser. No.10/637,847 filed Aug. 7, 2003, now U.S. Pat. No. 7,244,402 which claimspriority of U.S. provisional patent application No. 60/447,157 filedFeb. 12, 2003, and of U.S. provisional patent application No. 60/433,160filed Dec. 13, 2002. In addition, U.S. patent application Ser. No.10/637,847 is a continuation-in-part of U.S. patent application Ser. No.10/265,473, filed Oct. 4, 2002, now U.S. Pat. No. 7,306,672 which is inturn a continuation-in-part of U.S. patent application Ser. No.10/117,978 filed Apr. 5, 2002, now U.S. Pat. No. 7,195,670 which claimspriority of U.S. provisional patent application No. 60/323,524 filedSep. 17, 2001. Further, U.S. patent application Ser. No. 10/117,978 is acontinuation-in-part of U.S. application Ser. No. 09/887,997 filed Jun.22, 2001, now U.S. Pat. No. 7,052,545 which in turn is acontinuation-in-part of U.S. patent application Ser. No. 09/826,583filed Apr. 6, 2001, now U.S. Pat.No. 6,899,137 which is in turn acontinuation-in-part of U.S. patent application Ser. No. 09/724,784filed Nov. 28, 2000, now U.S. Pat. No. 7,144,616 which is in turn acontinuation-in-part of U.S. patent application Ser. No. 09/605,520,filed Jun. 27, 2000, now U.S. Pat. No. 7,601,270. U.S. patentapplication Ser. No. 09/605,520 claims priority of U.S. provisionalpatent application No. 60/186,856 filed Mar. 3, 2000, U.S. provisionalpatent application No. 60/147,199 filed Aug. 3, 1999, and U.S.provisional patent application No. 60/141,503 filed Jun. 28, 1999. Eachof these prior patent applications are hereby incorporated by referencefor all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Work described herein has been supported, in part, by NSF (xyz in a chipprogram); National Institute of Health grant CA 77373; NSERC (JuliePayette Fellowship); David H. & Lucille M. Packard Foundation; and G.Harold and Leila Y. Mathers Charitable Foundation. Work described hereinhas also been supported in part by National Science Foundation Grant No.CTS 0088649, National Institutes of Health Grant Nos. CA 77373 and HG1642-02, and Army Research Office Grant No. DAAD19-00-1-0392 awarded byDARPA. The United States Government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

Crystallization is an important technique to the biological and chemicalarts. Specifically, a high-quality crystal of a target compound can beanalyzed by x-ray diffraction techniques to produce an accuratethree-dimensional structure of the target. This three-dimensionalstructure information can then be utilized to predict functionality andbehavior of the target.

In theory, the crystallization process is simple. A target compound inpure form is dissolved in solvent. The chemical environment of thedissolved target material is then altered such that the target is lesssoluble and reverts to the solid phase in crystalline form. This changein chemical environment typically accomplished by introducing acrystallizing agent that makes the target material is less soluble,although changes in temperature and pressure can also influencesolubility of the target material.

In practice however, forming a high quality crystal is generallydifficult and sometimes impossible, requiring much trial and error andpatience on the part of the researcher. Specifically, the highly complexstructure of even simple biological compounds means that they are notamenable to forming a highly ordered crystalline structure. Therefore, aresearcher must be patient and methodical, experimenting with a largenumber of conditions for crystallization, altering parameters such assample concentration, solvent type, countersolvent type, temperature,and duration in order to obtain a high quality crystal, if in fact acrystal can be obtained at all.

Accordingly, there is a need in the art for methods and structures forperforming high throughput screening of crystallization of targetmaterials.

SUMMARY OF THE INVENTION

The use of microfluidic structures enables high throughput screening ofprotein crystallization. In one embodiment, an integrated combinatoricmixing chip allows for precise metering of reagents to rapidly create alarge number of potential crystallization conditions, with possiblecrystal formations observed on the chip. In an alternative embodiment,the microfluidic structures may be utilized to explore phase spaceconditions of a particular protein crystallizing agent combination,thereby identifying promising conditions and allowing for subsequentfocused attempts to obtain crystal growth.

An embodiment of a method in accordance with the present invention ofcrystallization, comprises, utilizing a microfludic formulator device togenerate a solubility fingerprint of a crystallization target over arange of conditions, and utilizing the microfluidic formulator to mapphase space around those conditions of the solubility fingerprintresulting in precipitation of the crystallization target.

Another embodiment of a crystallization method in accordance with thepresent invention, comprises, empirically determining a solubility curvefor a crystallization target mixed with a precipitant utilizing amicrofluidic device, and mixing the crystallization target with theprecipitant at a ratio that places a final concentration of thecrystallization target and the precipitant on a boundary of thesolubility curve.

An embodiment of an apparatus in accordance with the present inventionfor investigating crystallization, comprises, a microfluidic formulatorcomprising a microfluidic chamber configured to receive acrystallization target and a precipitant, a light source configured toilluminate the microfluidic chamber, and a light detector configured toreceive light transmitted through the microfluidic chamber.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIG. 7H is a front sectional view showing the valve of FIG. 7B in anactuated state.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a front sectional view of the valve of FIG. 7B showingactuation of the membrane.

FIG. 11 is a front sectional view of an alternative embodiment of avalve having a flow channel with a curved upper surface.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 17A shows a plan view of one embodiment of a combinatoric mixingdevice in accordance with the present invention.

FIG. 17B shows an enlarged plan view of a portion of the combinatoricmixing device of FIG. 17A.

FIG. 18 plots injection volume for a number of injection slugs.

FIG. 19 plots concentration for a number of injection slugs.

FIG. 20A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 20C shows the alignment of the first layer of elastomer of FIG. 20Awith one set of control channels in the second layer of elastomer ofFIG. 20B.

FIG. 20D also shows the alignment of the first layer of elastomer ofFIG. 20A with the other set of control channels in the second layer ofelastomer of FIG. 20B.

FIG. 21 shows a plan view of one embodiment of a rotary mixing structurein accordance with the present invention.

FIG. 22 shows a plan view of an array of combinatoric mixing structuresin accordance with the present invention.

FIG. 23 shows a enlarged view of the array of combinatoric mixingstructures of FIG. 22.

FIG. 24 shows a phase space of a mixture of protein and precipitatingagent.

FIG. 25 shows a hysteresis effect in a phase space of a mixture ofprotein and precipitating agent.

FIGS. 26A-26D show plan views illustrating operation of one embodimentof a cell pen structure in accordance with the present invention.

FIGS. 27A-27B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 28A-28D show plan views of operation of a structure utilizingcross-channel injection in accordance with the embodiment of the presentinvention.

FIGS. 29A-D are simplified schematic diagrams plotting concentrationversus distance for two fluids in diffusing across a microfluidic freeinterface in accordance with an embodiment of the present invention.

FIGS. 30A-B show simplified cross-sectional views of the attemptedformation of a macroscopic free-interface in a capillary tube.

FIGS. 31A-B show simplified cross-sectional views of convective mixingbetween a first solution and a second solution in a capillary tuberesulting from a parabolic velocity distribution of pressure drivenPoiseuille flow

FIGS. 32A-C show simplified cross-sectional views of interaction in acapillary tube between a first solution having a density greater thanthe density of second solution.

FIG. 33A shows a simplified cross-sectional view of a microfluidic freeinterface in accordance with an embodiment of the present invention.

FIG. 33B shows a simplified cross-sectional view of a conventionalnon-microfluidic interface.

FIGS. 34A-D show plan views of the priming of a flow channel andformation of a microfluidic free interface in accordance with anembodiment of the present invention.

FIGS. 35A-E show simplified schematic views of the use of“break-through” valves to create a microfluidic free interface.

FIG. 36A shows a simplified schematic view of a protein crystal beingformed utilizing a conventional macroscopic free interface diffusiontechnique.

FIG. 36B shows a simplified schematic view of a protein crystal beingformed utilizing diffusion across a microfluidic free interface inaccordance with an embodiment of the present invention.

FIG. 37A shows a simplified plan view of a flow channel overlapped atintervals by a forked control channel to define a plurality of chambers(A-G) positioned on either side of a separately-actuated interface valve.

FIGS. 37B-D plot solvent concentration at different times for the flowchannel shown in FIG. 37A.

FIG. 38A shows three sets of pairs of chambers connected bymicrochannels of a different length.

FIG. 38B plots equilibration time versus channel length.

FIG. 39 shows four pairs of chambers, each having different arrangementsof connecting microchannel(s).

FIG. 40A shows a plan view of a simple embodiment of a microfluidicstructure in accordance with the present invention.

FIG. 40B is a simplified plot of concentration versus distance for thestructure of FIG. 40A.

FIG. 41 plots the time required for the concentration in one of thereservoirs of FIG. 40 to reach 0.6 of the final equilibrationconcentration, versus channel length.

FIG. 42 plots the inverse of the time required for the concentration inone of the reservoirs to reach 0.6 of the final equilibrationconcentration (T_(0.6)), versus the area of the fluidic interface ofFIG. 40.

FIG. 43 presents a phase diagram depicting the phase space betweenfluids A and B, and the path in phase space traversed in the reservoirsas the fluids diffuse across the microfluidic free interface of FIG. 40

FIG. 44 shows an enlarged view of one embodiment of a chip holder inaccordance with the present invention.

FIG. 45A shows a simplified plan view of the alternative embodiment ofthe chip utilized to obtain experimental results.

FIG. 45B shows as simplified enlarged plan view of a set of threecompound wells of the chip shown in FIG. 45A.

FIG. 45C shows a simplified cross-sectional view of the compound wellsof FIGS. 45A-B.

FIG. 46 shows a plan view of the creation of a microfluidic freeinterface between a flowing fluid and a dead-ended branch channel.

FIG. 47 shows a simplified plan view of one embodiment of a microfluidicstructure in accordance with the present invention for creatingdiffusion gradients of two different species in different dimensions.

FIG. 48 shows a simplified plan view of an alternative embodiment of amicrofluidic structure in accordance with the present invention forcreating diffusion gradients of two different species in differentdimensions.

FIG. 49 shows a simplified plan view of a sorting device in accordancewith an embodiment of the present invention.

FIG. 50 plots Log(R/B) vs. number of slugs injected for one embodimentof a cross-flow injection system in accordance with the presentinvention.

FIG. 51 plots injected volume versus injection cycles during operationof one embodiment of a cross-flow injection structure in accordance withthe present invention.

FIG. 52 plots crystallization hits utilizing a microfluidic chip inaccordance with the present invention.

FIGS. 53A-B show plan and cross-sectional views of one embodiment of acrystal growing/harvesting chip in accordance with of one embodiment ofthe present invention.

FIG. 54A shows a plan view of one embodiment of a combinatoricmixing/storage structure in accordance with the present invention.

FIG. 54B shows an enlarged view of the mixing portion of the structureof FIG. 54A.

FIG. 54C shows an enlarged view of the storage array of the structure ofFIG. 54A.

FIG. 54D shows an enlarged view of one cell of the storage array of thestructure of FIG. 54C.

FIG. 55 plots macromolecule concentration versus solvent concentrationto define a portion of phase space of one macromolecule solventcombination.

FIGS. 56A-F are Gibbs free energy diagrams of various regions of thephase space shown in FIG. 55.

FIG. 57 plots trajectory through the phase space shown in FIG. 55achieved by a number of crystallization approaches.

FIG. 58A-D illustrates a schematic view of storage technique utilizing agated serpentine storage line.

FIG. 59 illustrates a schematic view of an embodiment wherein amultiplexer could be used to direct each experimental condition intoparallel storage channels.

FIG. 60 illustrates a schematic view of an embodiment wherein eachreaction condition is dead-end filled to the end of a storage line.

FIGS. 61A-F illustrate schematic views of an embodiment of a method inaccordance with the present invention for solubilizing a membraneprotein.

FIGS. 62A-D are simplified schematic diagrams illustrating positivedisplacement cross-injection (PCI) dispensing.

FIGS. 63A-D show photographs illustrating combinatoric mixing utilizinga microfluidic formulator.

FIG. 64A plots injected volume versus the number of PCI injections.

FIG. 64B plots the fraction of the ring of the combinatoric mixingstructure occupied versus the number of PCI injections.

FIG. 65A plots pixel standard deviation versus xylanase concentration.

FIG. 65B plots pixel standard deviation versus a number of chemicalconditions.

FIGS. 65C1-24 plot Xylanase phase for protein concentration versusconcentration of a first precipitant stock, under a variety of differentconditions.

FIG. 65D is a simplified schematic plot of xylanase phase space overdifferent relative protein/salt concentrations for on-chip andmicrobatch.

FIG. 66 is a simplified schematic plot of lysozymic phase space overdifferent relative protein/salt concentrations.

FIG. 67 is a histogram showing number of successful crystallizationconditions identified with sparse matrix screens (each at proteinconcentrations of 12 mg/mL and 23 mg/mL) and optimal screen.

FIG. 68 is a polarized micrograph of large single crystals growndirectly from optimal screen.

FIGS. 69A and 69B plot phase space behavior for different samples ofXylanase under microbatch conditions.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent application Ser. No. 09/826,585 filed Apr. 6, 2001, Ser. No.09/724,784 filed Nov. 28, 2000, and Ser. No. 09/605,520, filed Jun. 27,2000. These patent applications are hereby incorporated by reference.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm,150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogeneous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogeneous aspect, the bonding process usedto bind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure Polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thePolyisobutylene backbone, which may then be vulcanized as above.

Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:w=(BPb ⁴)/(Eh ³), where:  (1)

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21 a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebrospinal fluid, pressure present in the intra-ocularspace, and the pressure exerted by muscles during normal flexure. Othermethods of regulating external pressure are also contemplated, such asminiature valves, pumps, macroscopic peristaltic pumps, pinch valves,and other types of fluid regulating equipment such as is known in theart.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1 kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, close is expected to be smallerthan τopen. There is also a lag between the control signal and controlpressure response, due to the limitations of the miniature valve used tocontrol the pressure. Calling such lags t and the 1/e time constants τ,the values are: topen=3.63 ms, τopen=1.88 ms, tclose=2.15 ms,τclose=0.51 ms. If 3τ each are allowed for opening and closing, thevalve runs comfortably at 75 Hz when filled with aqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≧8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 10, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns. Inexperiments performed by the inventors, a pumping rate of 2.35 nL/s wasmeasured by measuring the distance traveled by a column of water in thin(0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30L and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log 2n) controllines.

8. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A to20D. FIG. 20A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 20is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 20C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 20D.

As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 20D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIGS. 20A-D allows a switchable flow array tobe constructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

9. Cell Pen/Cell Cage

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 26A-26D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 26A has been loaded with cells A-H that havebeen previously sorted. FIGS. 26B-26C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 26D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a.

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.27A and 27B show plan and cross-sectional views (along line 45B-45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 26A-26D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

The cross-flow channel architecture illustrated shown in FIGS. 26A-26Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 28A-E, which illustrate a plan view of mixingsteps performed by a microfabricated structures in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 28A, valve pair 7408 c-d is initially opened whilevalve pair 7408 a-b is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7404. Valve pair 7408 a-b is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 28B, valve pairs 7408 c-d are closed and 7408 a-bare opened, such that fluid sample 7410 is injected from intersection7412 into flow channel 7402 bearing a cross-flow of fluid. The processshown in FIGS. 28A-B can be repeated to accurately dispense any numberof fluid samples down cross-flow channel 7402.

While the embodiment of a process-channel flow injector structure shownin FIGS. 28A-B feature channels intersecting at a single junction, thisis not required by the present invention. Thus FIG. 28C shows asimplified plan view of another embodiment of an injection structure inaccordance with the present invention, wherein junction 7450 betweenintersecting flow channels 7452 is extended to provide additional volumecapacity. FIG. 28D shows a simplified plan view of yet anotherembodiment of an injection structure in accordance with the presentinvention, wherein elongated junction 7460 between intersecting flowchannels 7462 includes branches 7464 to provide still more injectionvolume capacity.

And while the embodiment shown and described above in connection withFIGS. 28A-28D utilizes linked valve pairs on opposite sides of the flowchannel intersections, this is not required by the present invention.Other configurations, including linking of adjacent valves of anintersection, or independent actuation of each valve surrounding anintersection, are possible to provide the desired flow characteristics.With the independent valve actuation approach however, it should berecognized that separate control structures would be utilized for eachvalve, complicating device layout.

FIG. 50 plots Log(R/B) vs. number of slugs injected for one embodimentof a cross-flow injection system in accordance with the presentinvention. The reproducibility and relative independence of metering bycross-flow injection from process parameters such as flow resistance isfurther evidenced by FIG. 51, which plots injected volume versus numberof injection cycles for cross-channel flow injection under a variety offlow conditions. FIG. 51 shows that volumes metered by cross-flowinjection techniques increase on a linear basis over a succession ofinjection cycles. This linear relationship between volume and number ofinjection cycles is relatively independent of flow resistance parameterssuch as elevated fluid viscosity (imparted by adding 25% glycerol) andthe length of the flow channel (1.0-2.5 cm).

10. Rotary Mixing Structure

Microfluidic control and flow channels in accordance with embodiments ofthe present invention may be oriented to rotary pump design whichcirculates fluid through a closed circuit flow channel. As used hereinthe term “closed circuit” has the meaning known in the art and refers toconfigurations that are circular and variations thereof such asellipsoids and ovals, as well as flow circuit paths having corners asare created by triangular, rectangular, or more complex shapes.

As illustrated in FIG. 21, a layer with flow channels 2100 has aplurality of sample inputs 2102, a mixing T-junction 2104, a centralcirculation loop 2106 (i.e., the substantially circular flow channel),and an output channel 2108. The overlay of control channels with a flowchannel can form a microvalve. This is so because the control and flowchannels are separated by a thin elastomeric membrane that can bedeflected into the flow channel or retracted therefrom.

The substantially circular central loop and the control channels thatintersect with it form the central part of the rotary pump. The pump(s)which cause solution to be flowed through the substantially circularflow channel consist of a set of at least three control channels 2110a-c that are adjacent to one another and which intersect thesubstantially circular branch flow channel 2106 (i.e., the centralloop).

When a series of on/off actuation sequences, such a 001, 011, 010, 110,100, 101, are applied to the control channels, the fluid in the centralloop can be peristaltically pumped in a chosen direction, eitherclockwise or counterclockwise. The peristaltic pumping action resultsfrom the sequential deflection of the membranes separating the controlchannels and flow channel into or out of the flow channel.

In general, the higher the actuation frequency, the faster the fluidrotates through the central loop. However, a point of saturation mayeventually be reached at which increased frequency does not result infaster fluid flow. This is primarily due to limitations in the rate atwhich the membrane can return to an unactuated position. As describedbelow in connection with the combination mixing device shown in FIGS.17A-B, various techniques may be adopted to maintain the connectionbetween faster activation and flow/mixing at higher frequencies.

While the system shown in FIG. 21 shows two sets of pumps (i.e., twosets of three control channels that overlay the substantially circularflow channel) a single pump can be utilized (i.e., a single set of threecontrol channels overlaying the substantially circular flow channel).Furthermore, while each pump is shown as including three controlchannels, a different number of control channels can be utilized, forexample, a single serpentine control channel having multiple cross-overpoints could be used.

A variety of different auxiliary flow channels which are in fluidcommunication with the central loop can be utilized to introduce andwithdrawn sample and reactant solutions from the central loop.Similarly, one or more exit or outlet flow channels in fluidcommunication with the central loop can be utilized to remove solutionfrom the central loop. For example, control valves can be utilized atthe inlet(s) and the outlet(s) to prevent solution flow into or out fromthe central loop.

Flow channel sizes and shapes can vary. With certain devices, thediameter of the channel tends to range from about 1 mm to 2 cm, althoughthe diameter can be considerably larger in certain devices (e.g., 4, 6,8, or 10 cm). Limits on how small the diameter of the circular flowchannel can be are primarily a function of the limits imposed by themultilayer soft lithography processes. Channel widths (either flow orcontrol) usually vary between 30 μm and 250 μm. However, channel widthin some devices is as narrow as 1 um. Channels of larger widths can alsobe utilized, but generally require some type of structural supportwithin the flow channel. Channel height generally varies between 5 and50 μm. In flow channels having a width of 100 μm or less, the channelheight may be 1 μm or smaller. The flow channel is typically rounded toallow for complete blockage of the channel once the membrane isdeflected into the channel. In some devices, the channels have shapessuch as octagons or hexagons. In certain devices, the flow channels arerounded and 100 μm wide and 10 μm high and control channels are 100 μmwide and 10 μm high. One system that has been utilized in certainstudies has utilized a central loop having a diameter of 2 cm, a flowchannel width of 100 μm and a depth of 10 μm.

While the channels typically have the foregoing sizes and shapes, itshould be recognized that the devices provided herein are not limited tothese particular sizes and shapes. For example, branches present in aclosed circuit flow channel may serve to control the dispersion andhence mixing of materials flowed therein.

II. Combinatoric Mixing

The various microfluidic elements described above can be combinedtogether to create a microfluidic device enabling accurate and rapidmixing of arbitrary combinations of input solutions on a microfluidicchip, thus enabling the creation of many thousands of differentsolutions from relatively few basic components.

1. Combinatoric Mixing Structure

FIG. 17A shows a plan view of one embodiment of a combinatoric mixingdevice in accordance with the present invention. FIG. 17B shows anenlarged view of one region of the combinatoric mixing device of FIG.17A.

Combination mixing device 1700 comprises flow channel network 1702comprising buffer import flow lines (BF₁-BF₁₆) and reagent input flowlines (RF₁-RF₁₆), which intersect at branched cross-flow injectorstructure 1704, which is in turn in fluid communication with rotarymixing structure 1706.

Control channel network 1710 comprises control lines C₁-C₂₄. Controllines C₁-C₈ interact with buffer input flow lines BF₁-BF₁₆ to createfirst multiplexer structure 1720 governing metering of buffer tocross-flow injector 1704. Control lines C₁-C₈ also interact with reagentinput flow lines RF₁-RF₁₆ to create second multiplexer structure 1722governing metering of reagent to cross-flow injector 1704.

Control lines C₉-C₁₁ interact with main flow channel 1750 to createfirst peristaltic pump 1752 responsible for flowing buffer into thecross-flow injection structure 1704. Reagent is flowed through inputflow lines RF₁-RF₁₆ under the influence of external pressure.

The right-most flow channel 1755 is controlled by a separate controlline (C₁₂) and is used to flush water/buffer past the multiplexer inletto avoid cross-contamination and subsequent insoluble salt formation inthe channels. Prior to this washing process, the pressure within controllines C₁-C₈ may be varied to provide a pumping action.

Specifically, by selectively actuating these lines it is possible topump only a selected channel, or to simultaneously pump all channelstogether. Furthermore, the pumping sequence may be designed to pump aspecified volume of fluid either forward or backward. Backward fluidpumping may be used to prevent the unwanted mixing of two fluidsbelonging to different lines.

One instance where backward fluid pumping may be important is to preventthe unwanted mixture of different soluble salts in adjacent lines toform insoluble salts blocking the flow channels. Such unwanted mixingmay be prevented in the following manner.

At the beginning of an experiment, buffer is pumped back into themultiplexer so that injected solutions are located a finite distancedownstream from the multiplexer inlet. After a channel containing afirst salt is selected from the multiplexer and the cross-injectionjunction has been flushed, a control line such as C₁₂ is released toflush water or buffer past the multiplexer outlet. This flushingeliminates most of the salt from the vicinity of the multiplexer outlet.However, small amounts of salt may have diffused from the nearby inlets.

Thus the multiplexer is then used to pump forward all the inlet linessimultaneously, causing any remaining salt solution to be swept away bythe buffer/water moving past the multiplexer outlet. The multiplexer isthen next used to pump backwards, so that fresh buffer is brought backinto all the lines. In this way buffer solution is present in each lineof the multiplexer until the line is selected and desired reagent isflushed through the selected line. This flow process ensures againstunwanted mixing of the reagents. Continuous operation for more than aweek using the flush/backflow method just described has shown thatincompatible salts, for example potassium phosphate and magnesiumchloride, may be used in adjacent lines without any unwanted mixing orformation of insoluble salts.

Control line C₁₃ gates the flow of reagent into cross-flow injector1704. Control lines C₁₄ and C₁₇ gate the flow into and out of rotarymixer 1706. Control lines C₁₅, C₁₆, and C₁₈ interact with rotary mixer1706 to form a third peristaltic pump responsible for creating thecircular flow within the mixer.

First outlet flow channel 1770 is in direct fluid communication withcross-flow injector 1704 and typically conveys waste material to firstoutlet 1790. Second outlet flow channel 1772 is in fluid communicationwith the cross-flow injector structure 1704 through rotary mixer 1706,and thus conveys waste material to second outlet 1792.

The combinatoric mixer shown in FIGS. 17A-B further includes aserpentine alternative outlet channel 1781 proximate to second outletflow channel 1772. The purpose of serpentine channel 1781 is as follows.Since the inlet, cross-junction, mixing ring, and outlet are in fluidicseries, the fluidic impedance is the sum of the component impedance. Ifthe impedance of the cross-injector is small compared to theinlet/outlet impedance, then changing the viscosity of the fluid in thecross-injective should have a minimal effect, thereby desirablyresulting in a decrease in metering sensitivity. For this reason,channel 1781 is long (with many bends) and of smaller diameter. Theoutlet for the combinatoric mixer is switched to the serpentine channel1781 during injection cycles.

Typical operation of the device shown in FIGS. 17A-B is as follows. Flowis directed horizontally through cross injection area 1704. Firstmultiplexer 1720 is used to select a flow line from buffer flow channelinputs (BF₁-BF₁₆) that flush through branched-cross injector 1704,through rotary mixer 1706, out second outlet channel 1772.

The flow of buffer through the device is then stopped by closing thevalves of peristaltic pump 1752, and flow is directed vertically throughthe cross-injector 1704. A reagent is then selected from the reagentflow channel inputs (RF₁-RF₁₆) using second multiplexer 1722. Thisreagent flushes through cross-injection area 1704 and then out throughfirst outlet channel 1770.

Flow is then once again directed horizontally through cross-injector1704, and the peristaltic pump 1752 is used to push an exact amount ofreagent into rotary mixing ring 1706. Every cycle of peristaltic pump1752 injects a well-defined volume (approximately 80 pL) into the rotarymixer 1706, so that the total amount injected into the ring may becontrolled by number of injection cycles.

Once the desired amount of the first reagent is injected into rotarymixer 1706, another reagent flow channel line (RF_(x)) is selected andthe injection process is repeated. In this way, arbitrary combinationsof the reagents may be introduced into the rotary mixer 1706. The rotarymixer has a total volume of 5 nL so that the rotary mixer mayaccommodate approximately 60 injection volumes.

Once the ingredients have been injected into the rotary mixer 1706,diffusive mixing occurs by Poiseuille flow resulting from peristalticpumping of the mixture around mixer 1706. Once mixing is complete, themixture is flowed through flow channel 1711 to second outlet channel1772, which can be in fluid communication with another region of thechip or another chip entirely (neither of which is shown in FIGS. 17A-B)for storage, analysis and/or further processing. These steps may berepeated for serial processing.

The specific embodiment of the combinatoric mixing device 1700 shown inFIGS. 17A-B also includes sample port 1780 in fluid communication withrotary mixer 1706 through flow channel 1782 in pressure communicationwith the peristaltic pump 1784 defined by the presence of controlchannels C₁₉₋₂₁. If desired, a sample, may be introduced through thesample input port and then injected into the mixer using peristalticpump. As described at length below, one potential application utilizingthis sample injector is to conduct high throughput mapping of phasespace by precipitation.

The above described mixing chip may be used on its own or incorporatedas key component in a larger microfluidic device. The chip may be usedto mix and meter arbitrary combinations of fluids that can be deliveredto downstream measurement or storage systems. By adding storage ormemory elements this mixing functionality allows for large scalescreening and processing of samples. For example, as discussed below,the outlet of the ring may be used to serially mix reagents and thensend them to fill an array of several thousand reaction chambers forstorage/screening purposes.

The combintoric mixing structure described prepares a mixed volume ofabout 5 nL. However, embodiments in accordance with the presentinvention are not limited to mixing at this or any other volume. Mixingvolumes achievable by microfluidic devices may range from over 1 μL, tobetween about 1 μL and 100 pL. The mixing volumes utilized forcrystallization studies may thus include 1 μL or less, 100 nL or less,10 nL or less, 1 nL or less, or 100 pL or less.

While the above description relates to serial implementation ofcombinatoric mixing, parallel implementations of the basic mixingelements are also possible. For example, an array of multiple fluidicstructures such as that shown in FIGS. 17A-B may be incorporated onto asingle chip

For example, FIG. 22 shows an overall plan view, and FIG. 23 shows anenlarged view, of a column of combinatoric mixers in accordance with anembodiment of the present invention. Combinatoric mixing device 2200comprises flow channel network 2202 including buffer input flow channelsBF₁-BF₁₀ controlled by the peristaltic pump structures 2204 formed bythe overlay of control channels C₂-C₄. Buffer input flow channelsBF₁-BF₁₀ are in fluid communication with mixing structures 2208 throughrespective branched cross-channel injectors 2210. Reagents selected fromreagent flow lines RF₁-RF₁₆ may be selected utilizing the multiplexer2212 created from the overlap of control channel network C₁, and thenflowed into cross-flow injectors 2210.

The overlap of control channels C₇, C₈, and C₁₀ over the closed circuitmixing structure defines peristaltic mixing pumps 2214. The overlap ofcontrol lines C₆ and C₉ create respective gate valves 2216 for themixing structures. Materials outlet from the mixing structures flowsthrough outlet lines 2218 for disposal.

Macromolecule samples may be injected from sample inlet lines 2220 incommon fluid communication with sample reservoir 2222, specificallyutilizing peristaltic pumping structures defined by the overlap ofcontrol lines C₁₁-C₁₃. Once macromolecule samples have been injectedinto the mixing structure, the formation of the solid phase can bemonitored by optical interrogation, utilizing a common light source anda bank of detectors appropriately positioned proximate to the mixingstructures. Alternatively, a plurality of mixing structures on the chipmay be scanned over a single detector utilizing a motorized stage.

As shown in FIGS. 22-23, the control lines for each mixing component maybe connected so that the entire array of mixing elements may be operatedwith no increase in control complexity. In one embodiment, all thebuffers may be used at the same time with identical reagents.Alternatively, every ring may be prepared in an identical fashion butwith a different sample. Thus, a parallel architecture would allow forthe simultaneous screening of one sample against the same reagents, butwith different buffers (having different pH), or the screening of manydifferent samples against identical conditions. In this way thethroughput of this system can be increased proportionally to the degreeof parallelization.

For example, a combinatoric mixing structure in accordance with anembodiment of the present invention is currently able to performapproximately 3000 protein solubility assays per day, so that the designof FIGS. 22-23, having 10 parallel mixing structures, can perform 30,000experiments per day. Each of these experiments requires an average of 1nL of protein sample so that a total of 100,000 experiments can beconducted in a little over three days using a total volume ofapproximately 100 uL of protein sample.

Another method of increasing throughput is to couple the combinatoricmixing structure to another fluidic structure that is designed toperform a fixed mixing function. For example, the combinatoric mixingstructure can be coupled via a multiplexer to a fluidic mixing matrix.The combinatoric mixing structure can be used to fill the N rows of thematrix with unique solutions, while the columns are connected to Ndifferent samples. In this way N mixing operations may be used to createN² unique reactions.

In one approach, such a combinatoric mixing chip could be placed intofluid communication with a flow channel pattern suitable for performingthe polymerase chain reaction (PCR). Alternatively, a fluidic structuremay be designed to allow for a broad range of mixing ratios to besimultaneously implemented based on geometric metering schemespreviously described.

The combinatoric mixing design has been implemented and used todemonstrate ultra-precise metering of a wide range of fluids havingdifferent physical properties (ionic strength, pH, viscosity, surfacetension). It has been determined that this metering and mixing system isextremely accurate, robust, and insensitive to the fluid properties.Fluid may be injected into the ring in volume increments ofapproximately 80 pL and with less than 1% error. The system is able tometer fluids with viscosities ranging from 1 to 400 cP with only a 5%variation in injected volume.

The speed of metering fluids through channels of the embodiments ofmicrofluidic structures in accordance with embodiments of the presentinvention is well approximated by Equation (2) below, which describesthe volume flux through a channel of circular cross-section:

$\begin{matrix}{{{Q = \frac{\pi\; a^{4}\Delta\; P}{8\mu\; L}};}{{where}\text{:}}{Q = {{volume}\mspace{14mu}{flux}\mspace{14mu}{in}\mspace{14mu}{channel}\mspace{14mu}\left( {{vol}\text{/}s} \right)}}{{a = {{dimension}\mspace{14mu}{of}\mspace{14mu}{channel}}};}{{L = {{length}\mspace{14mu}{of}\mspace{14mu}{channel}}};}{{{\Delta\; P} = {{changed}\mspace{14mu}{pressure}\mspace{14mu}{within}\mspace{14mu}{channel}}};}{and}{\mu = {{viscosity}\mspace{14mu}{of}\mspace{14mu}{actuation}\mspace{14mu}{fluid}\mspace{14mu}{within}\mspace{14mu}{{channel}.}}}} & (2)\end{matrix}$

Equation (2) essentially describes the ability to effect a change involume of fluid in a microfluidic channel. Where the fluid is actuationfluid and the microfluidic channel is a control channel, this volumeflux dictates the rate at which material may be flowed through a flowchannel adjacent to the control channel.

Per Equation (2), one way of achieving more rapid actuation/mixing is toincrease the dimension (a) of the control channel. In the embodiment ofthe combinatoric mixing device of FIGS. 17A-B, this has beenaccomplished by enlarging dimensions of the control channels to reducethe flow resistance of volumes of actuating fluid moving in and out ofthe actuated/deactuated control channels, respectively. Specifically,the height dimension of the control channels has been increased to 30 μmfrom 10 μm

Another way of achieving more rapid actuation is to raise the baseline(ground) pressure above atmospheric. Specifically, the rebound of theactuated membrane may be a rate limiting factor since the pressuredriving retraction may only be the channel pressure which typically isnear atmospheric. The rebound thus may often be solely due to theelastic properties of the membrane. By raising the ground pressure, inaccordance with embodiments of the present invention, the membrane mayalso back up into the control channel by the flowed fluid. In certainembodiments, the base pressure is around 6 psi, however the higher thisbaseline pressure, the faster the valve response.

Further per Equation (2), still another way of achieving more rapidactuation is to reduce the viscosity (μ) of the actuation fluid.Generally, water having a viscosity of 0.001 kg/m·s, or air having aviscosity of 0.0000018 kg/m·s, may be utilized as the actuation fluid.Water rather than air has been used as the actuation fluid due to thedesire to avoid the formation of air bubbles in the flow channels.However, the embodiment of the combinatoric mixing device shown in FIGS.17A-B utilizes air-filled control channels to increase actuation speed.Moreover, it has been discovered that the use of an elevated baselinepressure substantially reduces the incidence of bubble formation.

To summarize, combination of 1) larger peripheral control channeldimensions, 2) elevated baseline pressure, and 3) the use of air as anactuation fluid, has increased the maximum frequency of actuation ofvalves of the microfluidic combinatoric mixing device of FIGS. 17A-Bfrom about 10 Hz to about 100 Hz, with a resulting frequency of movementof fluid through the closed circuit mixer of about 4 Hz.

The metering and mixing performed by the combinatoric mixing chip wasfound to be correspondingly fast. A single mixing configuration iscapable of processing 3000 samples per day. By the parallel integrationof 20 such mixers on a single chip it is possible to processapproximately 60,000 reactions a day, making this device suitable forhigh throughput screening applications.

FIGS. 18 and 19 below show the results of some metering experimentsperformed on chip. FIG. 18 plots injection volume versus injected slugnumber for titration of 2 mM Bromphenol Blue (0.1 M Tus-HCl @ pH 8.1).Absorption measurements of FIG. 18 evidence the precision andrepeatability of metering. Each of the nine clusters consists of onehundred independent metering experiments conducted over the period often hours.

FIG. 19 plots concentration versus injected slug number for samplesexhibiting four different viscosities, as summarized in the followingTABLE 1.

TABLE 1 GLYCEROL VISCOSITY SLUG VOL. SAMPLE (%) (cP) (pL) R2 A 0 1 90.720.999 B 76 40 88.2 0.997 C 84 100 84.84 0.998 D 92 400 86.52 0.998The narrow deviation evidenced by the injected volumes in FIG. 19evidences absorption measurements showing the insensitivity of meteringto fluid properties, and in particular to a wide range of viscosities.

The combinatoric mixing device may find a variety of applications as aformulation tool to address problems in biology, chemistry, chemicalengineering and so forth in which it is necessary to find the optimalcombination of components in a recipe. This device chip and variantsthereof can be used to systematically screen through many variations inthe parameters of these recipes, thus providing a quick and inexpensivemeans to optimize recipes and formulations. Potential fields of useinclude microbiology, chemical synthesis, high throughput screening,drug discovery, medical diagnostics, pathogen identification, andenzymatic reactions (including but not limited to the polymerase chainreaction and all of its variants). The device can also serve toformulate a variety of lotions, creams, or food products, chemicalsynthesis, and so forth.

Embodiments of microfluidic structures in accordance with the presentinvention may be employed for applications as are more completelydescribed in PCT application PCT/US01/44869, filed Nov. 16, 2001 andentitled “Cell Assays and High Throughput Screening”, herebyincorporated by reference for all purposes. Examples of microfluidicstructures suitable for performing such applications include thosedescribed herein, as well as others described in U.S. nonprovisionalpatent application Ser. No. 10/118,466, “Nucleic Acid AmplificationUtilizing Microfluidic Devices”, filed Apr. 5, 2002, hereby incorporatedby reference for all purposes.

One promising application for the combinatoric device shown anddescribed in connection with FIGS. 17A-B is the solubilization ofmembrane proteins.

Membrane proteins are typically expressed in eukaryotic cells, wherethey are incorporated within the cell membranes. The three-dimensionalstructure of these membrane proteins can be determined by x-raydiffractometry of them in crystalline form.

Before such crystals can be formed, however, it is first often necessaryto stabilize the proteins in solution with the three-dimensional foldedshape that they possess when incorporated into the cell membrane. Thisis typically accomplished by addition of a detergent, which encloses themembrane protein in a small envelope of amphiphilic molecules (thedetergents) that emulate the environment of the cell membrane andprevent denaturation. Solubilizing the membrane proteins in this mannertypically requires experimentation with different buffers at differentpH values, ionic strengths, and including different detergents. Thesolubilization of membrane proteins is thus at heart a formulationproblem.

Since the membrane proteins are typically available to the researcher inonly small quantities, it is desirable to perform solubility studies insmall volumes, and in an automated fashion. Accordingly, thecombinatoric mixing device just described is well-suited to this task.

FIGS. 61A-G are schematic views of an assay by which screeningconditions may be assessed for success in solubilizing membraneproteins. This technique is appropriate for solubilization of membraneproteins for which a corresponding receptor or antibody is known.

FIG. 61A shows a first step of the method, wherein cells 6100 in testtube 6102 are lysed centrifuged to concentrate membranes containing themembrane proteins in a pellet 6104 at a bottom of the test tube. Pellet6104 is referred to as the lysate.

FIG. 61B shows a second step of the method, wherein membrane proteins6106 within the lysate are tagged with histadine (HIS) 6108 and a samplecontaining the HIS-tagged membrane proteins 6110 is flowed intomicrofluidic inlet channel 6112 into closed circuit mixing structure6114. FIG. 61C shows a third step, wherein mixture 6115 of detergent,buffers, and salts are injected into the mixing structure 6114 and mixedwith the HIS-tagged membrane proteins 6110 to solubilize the membraneprotein. At this stage the solubilized membrane proteins may or may notexhibit the folded three-dimensional shape exhibited in the cellmembrane.

FIG. 61D shows a fourth step, wherein the solubilized membrane proteinmixture is flowed out of the mixing structure 6114 through multiplexer6119 to first flow channel 6116 a over a nickel substrate 6118 toimmobilize the proteins. FIG. 61E shows a fifth step, wherein afluorescently tagged ligand or antibody 6118 is flowed over theimmobilized HIS-tagged protein sample 6110. The corresponding taggedligand/antibody will bind only to those immobilized proteins exhibitingthe same folded shape as in the cell membrane.

As shown in FIG. 61F, the flow channel containing the nickel is washedand a fluorescence measurement taken by irradiating the contents ofchannel 6118 from source 6150 and detecting at detector 6152 theresulting emitted fluorescence. Intensity of the detected fluorescentsignal may reveal the number of solubilized proteins exhibiting desirednaturally-occurring folded three-dimensional shape that allows bindingof the complementary fluorescent ligand/antibody. Subsequent variationof the solubilization conditions can optimize the number of proteinmolecules entering solution with the desired three-dimensional foldedshape. Optimization of the solubilization process can be achieved bycomparing the magnitude of signals detected from mixtures havingdifferent concentrations/identities of amphiphilic moieties or othersolution components.

At the conclusion of one solubilization process, the closed circuitmixing structure may be washed with a low pH buffer to elute the boundand labeled protein, and another solubilization experiment conducted. Ifa fresh nickel surface is needed, the next mixture can be directed fromthe mixing structure to another flow channel by the multiplexer.

While the embodiment illustrated and discussed in connection with FIGS.61A-F above involves exposing the solubilized membrane protein to thecomplementary detectable ligand after mixing in the closed circuitmicrofluidic mixing structure, this is not required by the presentinvention. In accordance with an alternative embodiment, thecomplementary ligand/antibody may be mixed directly with the taggedprotein in the mixing structure. This alternative embodiment may offermore accurate results where binding of the tagged protein to the nickelsubstrate may inhibit binding between the protein and its complementaryligand.

And while the embodiment illustrated and discussed in connection withFIGS. 61A-F above involves immobilizing the solubilized protein on aplanar nickel substrate, this is also not required by the presentinvention. Alternative embodiments could utilize a flow channel packedwith beads having surfaces exhibiting the desired immobilizationfunctionality.

And while the embodiment illustrated and discussed in connection withFIGS. 61A-F above involves the use of a ligand detectable by itsfluorescent properties, this is also not required by the presentinvention. Alternative embodiments could utilize radio-type ligandsdetectable utilizing a PET detector.

Another particularly promising application for the combinatoric deviceshown and described in connection with FIGS. 17A-B is crystallization ofmacromolecules, for example the membrane proteins whose solubilizationwas just discussed. Such crystallization requires the large scalescreening of many different reagents against a concentrated and purifiedprotein or macromolecule sample. Since protein is generally difficult toobtain and purify in large quantities, it is of the utmost importance tominimize sample consumption in screening trials. While conventionalmeans require microliter sample volumes per assay, the current inventionis capable of realizing sub-nanoliter reaction volumes.

Furthermore, since many thousands of unique solutions may be mixeddirectly on chip, the present invention may be used to do exhaustivescreening of protein crystallization conditions. This screening may bedone in a random or systematic way. Once mixed, crystallizationreactions may be routed to a locations device for storage andinspection, for example as is described in detail below.

2. Storage Structures

Combining the basic metering and mixing functionality of thecombinatoric mixing structure with a fluidic storage structure, allowsfor a complete protein crystallization workstation to be implemented onchip. In this way a researcher may explore the solubility of a proteinin various chemistries, decide which are the most promisingcrystallization conditions, and then set and incubate reactions forcrystal growth. In this way, screening, phase space exploration,optimization, and incubation may be achieved on a single microfluidicworkstation. A non-exclusive list of possible methods of storage isprovided below.

In accordance with one embodiment of the present invention, reactionsmay be stored by pumping pre-mixed reagent (crystallizing agents,additives, cryo-protectants . . . , sample) into a storage channel andseparating the experiments by an immiscible fluid (eg. Paraffin oil).FIGS. 58A-C are schematic views of one implementation of this storagetechnique.

In FIG. 58A, a sample is flowed into circular mixing device 5800 throughthe peristaltic pumping action of serial valves 5802 a-c. Mixture 5803is then created and flowed into cross-junction 5804. In FIG. 58B, thesample within cross-junction 5804 is routed to serpentine, storagechannel 5806 having end 5806 a controlled by valve 5810. In FIG. 58C,the valve configuration is changed, and an inert separating fluid 5808such as oil is flowed into cross-junction 5804. In FIG. 58D, theseparating fluid 5808 is flowed into storage channel 5806. The cycleillustrated in FIGS. 58A-D may then be repeated to provide a new samplevolume within storage line 5806. Samples stored within channel 5806 maybe recovered through end 5806 a through gate valve 5810.

Assuming that the storage channel has dimensions 100 μm wide*100 μmtall, a 1 nL sample would fill a length of channel equal to 100 μm.Assuming that the channel is serpentine and that adjacent legs areseparated by 100 μm, the total length of channel that would fit on a 1cm square storage area is approximately 1 cm*100/2=0.5 m. This wouldallow the storage of 0.5 m/100 μm=5000 reactions.

Since the entire length of fluid must be advanced for every addition, itmay prove difficult to pump this long length of fluid. To avoid thisproblem, FIG. 59 shows a schematic view of embodiment 5900 of areaction/storage scheme wherein multiplexer 5902 could be used to directan experimental sample and inert separating fluid into one of aplurality of parallel storage channels 5904.

In accordance with still another alternative embodiment, each reactioncould be dead-end filled to the end of the storage line so that theentire column of fluid need never be moved together. FIG. 60 shows asimplified schematic view of such an approach, wherein storage channel6000 is dead-ended.

A flow of air could be utilized to bias the samples and inert separatingliquid into the storage channel, with the air ultimately diffusing outof the channel through the elastomer material. In such an embodiment,the relatively high pressures required to accomplish dead-ended fillingcould be achieved using an external pressure source, thereby eliminatingthe need for a separate pump on the oil line. This dead-end fillingtechnique could be used to fill a single storage line as in theembodiment shown in FIGS. 58A-D, or many parallel storage lines througha multiplexer as in the embodiment shown in FIG. 59.

While FIGS. 58A-60 show the storage line as being integrated in a planarfashion as a channel on the chip, this is not required by the presentinvention. In accordance with alternative embodiments of the presentinvention the reagents may be off-loaded from the chip in the verticaldirection, for example into a glass capillary.

Still another approach for storing chemicals is to utilize diffusionassays. FIGS. 54A-D show a layout of a device that combines combinatoricmixing structure 5400 with an addressable storage array 5402. Array 5402allows for incubation on chip of 256 individual batch experiments or 128free interface diffusion experiments.

FIG. 54B shows a blow-up of the entrance to storage array 5402. Array5402 works on the dead-end loading principal discussed above. Once thereagents are mixed in ring 5406, they are pumped into serpentine channel5408 for temporary storage. Multiplexer 5410 at the storage array inletis then actuated to open one of the sixteen possible inlets, therebyselecting the array row to be addressed.

FIG. 54C shows an enlarged view of the storage array of FIG. 54A. FIG.54D shows an enlarged view of a single cell in the array of FIG. 54D.

Each row of the storage array has a control line 5452 that actuatesvalves 5454 separating storage chambers 5456 from the channel inlets,and a control line 5458 that separates the columns of the storage array.A single control line 5460 is further routed to every pair offluidically coupled chambers 5462 a-b to separate them until it isdesired to create a fluidic interface.

Once the row is selected by the multiplexer, the array column 5470 isselected by actuating a corresponding column valve 5472. In this way asingle chamber of the array is selected for filling.

Valves near the outlet of the ring are actuated to connect theserpentine storage line to the multiplexer inlet, and the storedsolution is pushed back out of the serpentine storage line and into themultiplexer area by pressurized air. This pressurization drives thefluid into the appropriate row of the storage array, pressurizing theair ahead of it and causing it to diffuse into the polymer.

While the chamber inlet valves remain closed, the fluid does not enterthe chamber, but rather remains in the dead volume between themultiplexer and the storage array (or partly in this volume and partlyin the storage array channels). A new line of the multiplexer is thenselected and the column valve is temporarily opened to allow the new rowto be flushed with buffer as a precaution to avoid cross contamination.Since only one line of the multiplexer is open the other rows of thestorage array are held fixed.

This new row is then emptied by blowing air through it, preparing it forthe next solution. These steps are repeated until all rows are filledwith a unique solution.

The column valve is then actuated, and the inlet valves opened, and allrows are simultaneously pressurized. This drives the solutions intotheir respective chambers. This entire process can be repeated for everycolumn until the array is filled with solutions (potentially a differentsolution in every chamber).

If the interface valves are held closed, the array of FIGS. 54A-D willaccommodate 256 (8 columns×16 rows) batch reactions. In applicationssuch as protein crystallography where diffusion across a microfluidicfree interface is desired, the interface valves may be opened tocommence the reactions. Since all solutions have previously beenseparately mixed in the ring, the experimenter has control over thesolutions.

For example, free-interface diffusion experiments for crystallizationmay be conducted in which one or more of the following is varied:identity and/or initial concentration of the precipitating agent;identity and/or initial concentration of the crystallized species;identity and/or initial concentration of additives; and identity and/orinitial concentration of cryo-protectants. The ability to mix a host ofdifferent agents into small volumes of protein solution prior to freeinterface diffusion experiments offers an important advantage overconventional crystallization approaches, where typically a standardprotein stock is used against different crystallization agents. Themicrofluidic network described above thus offers a flexible platform forcrystallization.

The array of FIGS. 54A-D also allows for sample recovery, addressablewell flushing, and the reusing of reaction chambers. Specifically, torecover sample or flush a well the appropriate inlet valves are openedon the column that has the well to be emptied/flushed. One of the rowsof the multiplexer that connects one of the chambers of the pair to beflushed is selected.

The array row that connects the pair that was not opened at themultiplexer is then opened at the end of the array, and the row that wasopened at the multiplexer is closed at the end of the array.

This manipulation causes and open fluidic path through the selected rowof the multiplexer, through the chamber pair to be emptied/flushed, andout the row selected at the outlet. In this way a single chamber paircan be addressed and flushed.

III. Crystallization Structures and Methods

High throughput screening of crystallization of a target material, orpurification of small samples of target material by recrystallization,may be accomplished by simultaneously introducing a solution of thetarget material at known concentrations into a plurality of chambers ofa microfabricated fluidic device. The microfabricated fluidic device isthen manipulated to vary solution conditions in the chambers, therebysimultaneously providing a large number of crystallization environments.Control over changed solvent conditions may result from a variety oftechniques, including but not limited to metering of volumes of acrystallizing agent into the chamber by volume exclusion, by entrapmentof liquid volumes determined by the dimensions of the microfabricatedstructure, or by cross-channel injection into a matrix of junctionsdefined by intersecting orthogonal flow channels.

Crystals resulting from crystallization in accordance with embodimentsof the present invention can be utilized for x-ray crystallography todetermine three-dimensional molecular structure. Alternatively, wherehigh throughput screening in accordance with embodiments of the presentinvention does not produce crystals of sufficient size for direct x-raycrystallography, the crystals can be utilized as seed crystals forfurther crystallization experiments. Promising screening results canalso be utilized as a basis for further screening focusing on a narrowerspectrum of crystallization conditions, in a manner analogous to the useof standardized sparse matrix techniques.

Systems and methods in accordance with embodiments of the presentinvention are particularly suited to crystallizing larger biologicalmacromolecules or aggregates thereof, such as proteins, nucleic acids,viruses, and protein/ligand complexes. However, crystallization inaccordance with the present invention is not limited to any particulartype of target material.

As employed in the following discussion, the term “crystallizing agent”describes a substance that is introduced to a solution of targetmaterial to lessen solubility of the target material and thereby inducecrystal formation. Crystallizing agents typically includecountersolvents in which the target exhibits reduced solubility, but mayalso describe materials affecting solution pH or materials such aspolyethylene glycol that effectively reduce the volume of solventavailable to the target material. The term “countersolvent” is usedinterchangeably with “crystallizing agent”.

1. Crystallization by Volume Entrapment

FIG. 45A shows a simplified plan view of an embodiment of acrystallization system wherein metering of different volumes ofcountersolvent is determined by photolithography during formation of theflow channels. FIG. 45B shows a simplified enlarged plan view of a setof three compound wells of the device of FIG. 45A. FIG. 45C shows asimplified cross-sectional view of the wells of FIG. 45B along lineC-C′. This chip design employed metering of target solution andcrystallizing agent utilizing the volume entrapment technique.

Specifically, each chip 9100 contains three compound wells 9102 for eachof the 48 different screen conditions, for a total of 144 assays perchip. A compound well 9102 consists of two adjacent wells 9102 a and9102 b etched in a glass substrate 9104, and in fluidic contact via amicrochannel 9106 In each of the compound wells 9102, the proteinsolution is combined with the screen solution at a ratio that is definedby the relative size of the adjacent wells 9102 a-b. In the particularembodiment shown in FIGS. 45A-C, the three ratios were(protein:solution) 4:1, 1:1, and 1:4. The total volume of each assay,including screen solution, is approximately 25 nL. However, the presentinvention is not limited to any particular volume or range of volumes.Alternative embodiments in accordance with the present invention mayutilize total assay volumes of less than 10 nL, less than 5 nL, lessthan 2.5 nL, less than 1.0 nL, and less than 0.5 nL.

The chip control layer 9106 includes an interface control line 9108, acontainment control line 9110 and two safety control lines 9112. Controllines 9108, 9110, and 9112 are filled with water rather than air inorder to maintain a humid environment within the chip and to preventdehydration of the flow channels and chambers in which crystallizationis to be performed.

The interface valves 9114 bisect the compound wells 9102, separating theprotein from the screen until completion of loading. Containment valves9116 block the ports of each compound well 9102, isolating eachcondition for the duration of the experiment. The two safety valves 9118are actuated during protein loading, and prevent spillage of proteinsolution in the event of a failed interface valve.

Fabrication of the microfluidic devices utilized in the experiments wereprepared by standard multilayer soft lithography techniques and sealedto an etched glass slide by baking at 80° C. for 5 hours or greater. Theglass substrate is masked with a 16 um layer of 5740 photoresist, and ispatterned using standard photolithography. The glass substrate is thenetched in a solution of 1:1:1 (BOE:H₂O:2NHCl) for 60 minutes, creatingmicro-wells with a maximum depth of approximately 80 μm.

The chip fabrication protocol just described is only one example of apossible embodiment of the present invention. In accordance withalternative embodiments, the crystallization chambers and flow channelscould be defined between a planar substrate and a pattern of recessesformed entirely in the lower surface of the elastomer portion. Stillfurther alternatively, the crystallization chambers and flow channelscould be defined between a planar, featureless lower surface of theelastomer portion and a pattern of recesses formed entirely in thesubstrate.

Crystallization on chip is set up as follows. All control lines in chipcontrol layer 9106 are loaded with water at a pressure of 15-17 psi.Once the control lines are filled and valves 9114 and 9116 arecompletely actuated, the containment valve 9116 is released, and proteinis loaded through the center via 9120 using about 5-7 psi. The proteinsolution completely fills the protein side of each compound well 9102.Failed valves, if present, are then identified, and vacuum grease isplaced over the corresponding screen via to prevent subsequentpressurization, and possible contamination of the remaining conditions.2.5 to 4 μL of a sparse matrix screen (typically Hampton Crystal ScreenI, 1-48) are then pipetted into the screen vias 9122. The safety valves9118 are released, and a specially designed chip holder (describedbelow) is used to create a pressurized (5-7 psi) seal over all 48 screenvias 9122. The screen solutions are dead end loaded, filling the screenside of each compound well. Protein and crystal screen reagents are keptseparate with the interface valve until all wells are loaded, at whichpoint the containment valve is closed and the interface valve opened toallow diffusion between liquid volumes present in the two halves of thecompound wells 9102.

For these experiments, the average time spent setting up an experiment,including filling control lines, was approximately 35 min, with thefastest experiment taking only 20 minutes to set up. This set up timecould potentially be reduced even further through the use of roboticpipetting of solutions to the chip, or through the use of pressures toload and prime delivered solutions, or through use of a microfluidicmetering device, for example the combinatorial mixing structurepreviously described.

As previously illustrated, embodiments of microfluidic devices inaccordance with the present invention may utilize on-chip reservoirs orwells. However, in a microfluidic device requiring the loading of alarge number of solutions, the use of a corresponding large number ofinput tubes with separate pins for interfacing each well may beimpractical given the relatively small dimensions of the fluidic device.In addition, the automated use of pipettes for dispensing small volumesof liquid is known, and thus it therefore may prove easiest to utilizesuch techniques to pipette solutions directly on to wells present on theface of a chip.

Capillary action may not be sufficient to draw solutions from on-chipwells into active regions of the chip, particularly where dead-endedchambers are to be primed with material. In such embodiments, one way ofloading materials into the chip is through the use of externalpressurization. Again however, the small dimensions of the devicecoupled with a large number of possible material sources may renderimpractical the application of pressure to individual wells through pinsor tubing.

Accordingly, FIG. 44 shows an exploded view of a chip holder 11000 inaccordance with one embodiment of the present invention. Bottom portion11002 of chip holder 11000 includes raised peripheral portion 11004surrounding recessed area 11006 corresponding in size to the dimensionsof chip 11008, allowing microfluidic chip 11008 to be positionedtherein. Peripheral region 11004 further defines screw holes 11010.

Microfluidic device 11008 is positioned within recessed area 11006 ofbottom portion 11002 of chip holder 11000. Microfluidic device 11008comprises an active region 11011 that is in fluidic communication withperipheral wells 11012 configured in first and second rows 11012 a and11012 b, respectively. Wells 11012 hold sufficient volumes of materialto allow device 11008 to function. Wells 11012 may contain, for example,solutions of crystallizing agents, solutions of target materials, orother chemical reagents such as stains. Bottom portion 11002 contains awindow 11003 that enables active region 11011 of chip 11008 to beobserved.

Top portion 11014 of chip holder 11000 fits over bottom chip holderportion 11002 and microfluidic chip 11008 positioned therein. For easeof illustration, in FIG. 80 top chip holder portion 11014 is showninverted relative to its actual position in the assembly. Top chipholder portion 11014 includes screw holes 11010 aligned with screw holes11010 of lower holder portion 11002, such that screws 11016 may beinserted through holes 11010 secure chip between portions 11002 and11014 of holder 11000. Chip holder upper portion 11014 contains a window11005 that enables active region 11011 of chip 11008 to be observed.

Lower surface 11014 a of top holder portion 11014 includes raisedannular rings 11020 and 11022 surrounding recesses 11024 and 11026,respectively. When top portion 11014 of chip holder 11000 is pressedinto contact with chip 11008 utilizing screws 11016, rings 11020 and11022 press into the soft elastomeric material on the upper surface ofchip 11008, such that recess 11024 defines a first chamber over top row11012 a of wells 11012, and recess 11026 defines a second chamber overbottom row 11012 b of wells 11012. Holes 11030 and 11032 in the side oftop holder portion 11014 are in communication with recesses 11024 and11026 respectively, to enable a positive pressure to be applied to thechambers through pins 11034 inserted into holes 11030 and 11032,respectively. A positive pressure can thus simultaneously be applied toall wells within a row, obviating the need to utilize separateconnecting devices to each well.

In operation, solutions are pipetted into the wells 11012, and then chip11008 is placed into bottom portion 11002 of holder 11000. The topholder portion 11014 is placed over chip 11008, and is pressed down byscrews. Raised annular rings 11020 and 11022 on the lower surface of topholder portion 11014 make a seal with the upper surface of the chipwhere the wells are located. Solutions within the wells are exposed topositive pressures within the chamber, and are thereby pushed into theactive area of microfluidic device.

The downward pressure exerted by the chip holder may also pose theadvantage of preventing delamination of the chip from the substrateduring loading. This prevention of delamination may enable the use ofhigher priming pressures.

The chip holder shown in FIG. 44 represents only one possible embodimentof a structure in accordance with the present invention.

2. Control Over Other Factors Influencing Crystallization

While the above crystallization structures describe altering theenvironment of the target material through introduction of volumes of anappropriate crystallization agent, many other factors are relevant tocrystallization. Such additional factors include, but are not limitedto, temperature, pressure, concentration of target material in solution,equilibration dynamics, and the presence of seed materials.

In specific embodiments of the present invention, control overtemperature during crystallization may be accomplished utilizing acomposite elastomer/silicon structure previously described.Specifically, a Peltier temperature control structure may be fabricatedin an underlying silicon substrate, with the elastomer aligned to thesilicon such that a crystallization chamber is proximate to the Peltierdevice. Application of voltage of an appropriate polarity and magnitudeto the Peltier device may control the temperature of solvent andcountersolvent within the chamber.

Alternatively, as described by Wu et al. in “MEMS Flow Sensors forNano-fluidic Applications”, Sensors and Actuators A 89 152-158 (2001),crystallization chambers could be heated and cooled through theselective application of current to a micromachined resistor structureresulting in ohmic heating. Moreover, the temperature of crystallizationcould be detected by monitoring the resistance of the heater over time.The Wu et al. paper is hereby incorporated by reference for allpurposes.

It may also be useful to establish a temperature gradient across amicrofabricated elastomeric crystallization structure in accordance withthe present invention. Such a temperature gradient would subject targetmaterials to a broad spectrum of temperatures during crystallization,allowing for extremely precise determination of optimum temperatures forcrystallization.

With regard to controlling pressure during crystallization, embodimentsof the present invention employing metering of countersolvent by volumeexclusion are particularly advantageous. Specifically, once the chamberhas been charged with appropriate volumes of solvent and countersolvent,a chamber inlet valve may be maintained shut while the membraneoverlying the chamber is actuated, thereby causing pressure to increasein the chamber. Structures in accordance with the present inventionemploying techniques other than volume exclusion could exert pressurecontrol by including flow channels and associated membranes adjacent tothe crystallization chamber and specifically relegated to controllingpressure within the channel.

Another factor influencing crystallization is the amount of targetmaterial available in the solution. As a crystal forms, it acts as asink to target material available in solution, to the point where theamount of target material remaining in solution may be inadequate tosustain continued crystal growth. Therefore, in order to growsufficiently large crystals it may be necessary to provide additionaltarget material during the crystallization process.

Accordingly, the cell pen structure previously described in connectionwith FIGS. 27A-27B may be advantageously employed in crystallizationstructures in accordance with embodiments of the present invention toconfine growing crystals within a chamber. This obviates the danger ofwashing growing crystals down a flow channel that is providingadditional target material, causing the growing crystals to be lost inthe waste.

Moreover, the cell cage structure of FIGS. 27A-27B may also be usefulduring the process of crystal identification. Specifically, salts areoften present in the sample or countersolvent, and these salts may formcrystals during crystallization attempts. One popular method ofdistinguishing the growth of salt crystals from the target crystals ofinterest is through exposure to a staining dye such as IZIT™,manufactured by Hampton Research of Laguna Niguel, Calif. This IZIT™ dyestains protein crystals blue, but does not stain salt crystals.

However, in the process of flowing the IZIT™ dye to the crystallizationchamber holding the crystals, the crystals may be dislodged, swept away,and lost. Therefore, the cell pen structure can further be employed incrystallization structures and methods in accordance with the presentinvention to secure crystals in place during the staining process.

FIG. 49 shows an embodiment of a sorting device for crystals based uponthe cell cage concept. Specifically, crystals 8501 of varying sizes maybe formed in flow channel 8502 upstream of sorting device 8500. Sortingdevice 8500 comprises successive rows 8504 of pillars 8506 spaced atdifferent distances. Inlets 8508 of branch channels 8510 are positionedin front of rows 8504. As crystals 8501 flow down channel 8502, theyencounter rows 8504 of pillars 8506. The largest crystals are unable topass between gap Y between pillars 8506 of first row 8504 a, andaccumulate in front of row 8504 a. Smaller sized crystals are gatheredin front of successive rows having successively smaller spacings betweenpillars. Once sorted in the manner described above, the crystals ofvarious sizes can be collected in chambers 8512 by pumping fluid throughbranch channels 8510 utilizing peristaltic pumps 8514 as previouslydescribed. Larger crystals collected by the sorting structure may besubjected to x-ray crystallographic analysis. Smaller crystals collectedby the sorting structure may be utilized as seed crystals in furthercrystallization attempts.

Another factor influencing crystal growth is seeding. Introduction of aseed crystal to the target solution can greatly enhance crystalformation by providing a template to which molecules in solution canalign. Where no seed crystal is available, embodiments of microfluidiccrystallization methods and systems in accordance with the presentinvention may utilize other structures to perform a similar function.

For example, as discussed above, flow channels and chambers ofstructures in accordance with the present invention are typicallydefined by placing an elastomeric layer containing microfabricatedfeatures into contact with an underlying substrate such as glass. Thissubstrate need not be planar, but rather may include projections orrecesses of a size and/or shape calculated to induce crystal formation.In accordance with one embodiment of the present invention, theunderlying substrate could be a mineral matrix exhibiting a regulardesired morphology. Alternatively, the underlying substrate could bepatterned (i.e. by conventional semiconductor lithography techniques) toexhibit a desired morphology or a spectrum of morphologies calculated toinduce crystal formation. The optimal form of such a substrate surfacemorphology could be determined by prior knowledge of the targetcrystals.

Embodiments of crystallization structures and methods in accordance withthe present invention offer a number of advantages over conventionalapproaches. One advantage is that the extremely small volumes(nanoliter/sub-nanoliter) of sample and crystallizing agent permit awide variety of recrystallization conditions to be employed utilizing arelatively small amount of sample.

Another advantage of crystallization structures and methods inaccordance with embodiments of the present invention is that the smallsize of the crystallization chambers allows crystallization attemptsunder hundreds or even thousands of different sets of conditions to beperformed simultaneously. The small volumes of sample and crystallizingagent employed in recrystallization also result in a minimum waste ofvaluable purified target material.

A further advantage of crystallization in accordance with embodiments ofthe present invention is relative simplicity of operation. Specifically,control over flow utilizing parallel actuation requires the presence ofonly a few control lines, with the introduction of sample andcrystallizing agent automatically performed by operation of themicrofabricated device permits very rapid preparation times for a largenumber of samples with the added advantages of parsimonious use ofsample solutions, ease of set-up, creation of well defined fluidicinterfaces, control over equilibration dynamics, and the ability toconduct high-throughput parallel experimentation. These advantages aremade possible by a number of features of the instant invention.

Microfluidics enables the handling of fluids on the sub-nanoliter scale.Consequently, there is no need to use large containment chambers, andhence, assays may be performed on the nanoliter, or subnanoliter scale.The utilization of extremely small volumes allows for thousands ofassays to be performed to consume the same sample volume required forone macroscopic free-interface diffusion experiment. This reduces costlyand time-consuming amplification and purification steps, and makespossible the screening of proteins that are not easily expressed, andhence must be purified from a bulk sample.

Microfluidics further offers savings in preparation times, as hundreds,or even thousands of assays may be performed simultaneously. The use ofscaleable metering techniques as previously described, allow forparallel experimentation to be conducted without increased complexity incontrol mechanisms.

Still another advantage of crystallization systems in accordance withembodiments of the present invention is the ability to control solutionequilibration rates. Crystal growth is often very slow, and no crystalswill be formed if the solution rapidly passes through an optimalconcentration on the way to equilibrium. It may therefore beadvantageous to control the rate of equilibration and thereby promotecrystal growth at intermediate concentrations. In conventionalapproaches to crystallization, slow-paced equilibrium is achieved usingsuch techniques as vapor diffusion, slow dialysis, and very smallphysical interfaces.

However, crystallization in accordance with embodiments of the presentinvention allows for control over the rate of solution equilibrium. Insystems metering crystallizing agent by volume exclusion, the overlyingmembrane can be repeatedly deformed, with each deformation giving riseto the introduction of additional crystallizing agent. In systems thatmeter crystallizing agent by volume entrapment, the valves separatingsample from crystallizing agent may be opened for a short time to allowfor partial diffusive mixing, and then closed to allow chamberequilibration at an intermediate concentration. The process is repeateduntil the final concentration is reached. Either the volume exclusion orentrapment approaches enables a whole range of intermediateconcentrations to be screened in one experiment utilizing a singlereaction chamber. As discussed in detail below, control over kinetics ofthe crystallization process may be controlled by varying the length orcross-sectional area of a capillary connection between reservoirscontaining the sample and crystallizing agent, respectively.

The manipulation of solution equilibrium over time also exploitsdifferential rates of diffusion of macromolecules such as proteinsversus much smaller crystallizing agents such as salts. As large proteinmolecules diffuse much more slowly than the salts, rapidly opening andclosing interface valves allows the concentration of crystallizing agentto be significantly changed, while at the same time very little sampleis lost by diffusion into the larger volume of crystallizing agent.Moreover, as described above, many crystallization structures describedreadily allow for introduction of different crystallizing agents atdifferent times to the same reaction chamber. This allows forcrystallization protocols prescribing changed solvent conditions overtime. Temperature control over equilibration is discussed in detailbelow.

3. Analysis of Crystal Structure from Protein on Chip

The utility of the chip is ultimately dependent on its' ability toquickly generate high quality diffraction patterns at a reduced cost. Aclear path from the chip-to-protein structure is therefore invaluable.Several paths from in-chip crystals to diffraction data are discussedbelow.

One possible application for a chip is determination of favorablecrystallization conditions that can subsequently be reproduced usingconventional techniques. Correspondence between the chip and twoconventional techniques (micro batch and hanging drop) has been shown tobe variable (between 45% and 80%). However, this variation is not afeature unique to the chip. These widely used crystallization techniquesshow only marginal correspondence (e.g. 14 of 16 hanging drop hits forlysozyme do not occur in microbatch), and often show variation withinthemselves. As a tool for screening initial crystallization conditions,the chip may be able to identify as many promising conditions.

FIG. 52 shows a comparison of the number of hits generated on sixdifferent protein samples (lysozyme, glucose isomerase, proteinase K, Bsubunit of topoisomerase VI, xylanase, and bovine pancrease trypsin)using the three different technologies. In FIG. 52 only crystals,microcrystals, rods, and needles are counted as hits, while spherulitesand precipitation is not counted. The data on Proteinase K is a sum ofthe experiments with and without PMSF, and data for the B subunit oftopoisomerase VI has not been included for lack of hanging drop data(although the chip far outperformed micro-batch in this case).Inspection of FIG. 52 shows that in four of the six cases, the chipproduced more hits than either conventional method.

In order to understand differences between crystallization methods toidentify possible reasons for productivity of the chip, we mustappreciate that the three methods produce different thermodynamicconditions on both short and long time scales. In order to induceprotein crystallization, one must make the crystallization energeticallyfavorable (supersaturation condition), and maintain these conditionslong enough for crystal growth to occur.

There are also different degrees of supersaturation. In lowsupersaturation, crystal growth tends to be supported, while nucleationof new crystals is relatively unlikely. In high supersaturation,nucleation is rapid, and many small low quality crystals may often beformed. In the three methods considered here, the condition ofsupersaturation is achieved through the manipulation of the relative,and absolute, concentration of protein and counter-solvent.

A comparison of the phase space evolution/equilibration of the threemethods is shown in FIG. 57. For the micro batch technique, mixing ofthe two solutions is quick, and when kept under impermeable oil layers,little significant concentration occurs over time. Micro batch thereforetends to sample only a single point in phase space, maintainingapproximately the same condition over time.

Hanging drop starts out like micro batch, with rapid mixing of the twosolutions, but then undergoes a concentration on a longer timescale(hours to days) due to vapor equilibration with the more concentratedsalt/precipitant reservoir. During the evaporative dehydration of thedrop, the ratio of protein to precipitant remains constant.

As described in detail below in the description of Microfluidic FreeInterface Diffusion, on the short time scale the chip dynamics mostclosely resemble a free interface diffusion experiment. Mixing is slow,and the rate of species equilibration (protein/precipitant/proton/salt)depends on species' diffusion constants. Small molecules such as saltshave large diffusion constants, and hence equilibrate quickly. Largemolecules (e.g. proteins) have small diffusion constants, andequilibrate more slowly.

The crystallization technique of free interface diffusion in capillariesmay more closely emulate the chip results. Traditionally this method isnot often used due to the difficulty of reliably setting up awell-defined interface. However, in microfluidic environments it isrelatively easy to establish reliable free-interface diffusionexperiments. Additional discussion of the formation of microfluidicfree-interfaces is presented below. In another application of thecrystallization chip, crystals may be grown for harvesting usingconventional methods.

If high quality crystals can be grown in, and extracted from the chip,crystallization conditions need not be exported. Since the chip can beremoved from the glass substrate, it is also possible to extract proteincrystals.

As previously described, once a protein crystal has been formed,information about its three dimensional structure can be obtained fromdiffraction of x-rays by the crystal. However, application of highlyenergetic radiation to the protein tends to generate creates heat.X-rays are also ionizing, and can result in the production of freeradicals and broken covalent bonds. Either heat or ionization maydestroy or degrade the ability of a crystal to diffract incident x-rays.

Accordingly, upon formation of a crystal a cryogenic material istypically added to preserve the crystalline material in its alteredstate. However, the sudden addition of cryogen can also damage acrystal. Therefore, it would be advantageous for an embodiment of acrystallization chip in accordance with the present invention to enablethe direct addition of cryogen to the crystallization chamber once acrystalline material is formed therein.

In addition, protein crystals are extremely delicate, and can quicklycrumble or collapse in response to physical trauma. Accordingly,harvesting a crystal unharmed from the small chambers of a chip poses apotential obstacle to obtaining valuable information about thecrystalline material.

Therefore, it would also be advantageous for an alternative embodimentof a crystallization chip in accordance with the present invention toallow direct interrogation by x-ray radiation of crystalline materialsformed in a chip, thereby obviating entirely the need for separatecrystal harvesting procedures.

Accordingly, FIG. 53A shows a plan view of a simplified embodiment of acrystal growing chip in accordance with the present invention. FIG. 53Bshows a simplified cross-sectional view of the embodiment of the crystalgrowing chip shown in FIG. 53A along line B-B′.

Harvesting/growing chip 9200 comprises elastomer portion 9202 overlyingglass substrate 9204. Glass substrate 9204 computes three etched wells9206 a, 9206 b and 9206 c. Placement of elastomer portion 9202 overglass substrate 9204 thus defines three corresponding chambers in fluidcommunication with each other through flow channels 9208. The flow ofmaterials through flow channels 9208 is controlled by valves 9210defined by the overlap of control lines 9212 over control channels 9208.

During operation of growth/harvesting chip 9200, valves 9210 areinitially activated to prevent contact between the contents of chambers9206 a, 9206 b and 9206 c. Chambers 9206 a, 9206 b and 9206 c are thenseparately charged through wells 9214 with different materials foreffecting crystallization. For example, chamber 9206 a may be chargedwith a protein solution, chamber 9206 b may be charged with acrystallizing agent, and chamber 9206 c may be charged with a cryogen.

The first control line 9212 may then be deactivated to open valve 9210a, and thereby allowing diffusion of protein solution and crystallizingagent. Upon formation of a crystal 9216, the remaining control lines9212 may be deactivated to allow the diffusion of cryogen from chamber9206 c to preserve the crystal 9216.

Next, the entire chip 9200 may be mounted in an x-ray diffractionapparatus, with x-ray beam 9218 applied from source 9220 against crystal9216 with diffraction sensed by detector 9222. As shown in FIG. 53B, thegeneral location of wells 9206 corresponds to regions of reducedthickness of both elastomer portion 9202 and underlying glass portion9204. In this manner, radiation beam 9218 is required to traverse aminimum amount of elastomer and glass material prior to and subsequentto encountering crystal 9216, thereby reducing the deleterious effect ofnoise on the diffracted signal received.

While one example of a protein growth/harvesting chip has been describedabove in connection with FIGS. 53A-B, embodiments in accordance with thepresent invention are not limited to this particular structure. Forexample, while the embodiment of the current embodiment that isdescribed utilizes a glass substrate in which microchambers have beenetched, fabrication of microfluidic structures in accordance with thepresent invention is not limited to the use of glass substrates.Possible alternatives for fabricating features in a substrate includeinjection molding of plastics, hot embossing of plastics such as PMMA,or fabricating wells utilizing a photocurable polymer such as SU8photoresist. In addition, features could be formed on a substrate suchas glass utilizing laser ablation, or features could be formed byisotropic or aniosotropic etching of a substrate other than glass, suchas silicon.

Potential advantages conferred by alternative fabrication methodsinclude but are not limited to, more accurate definition of featuresallowing for more dense integration, and ease of production (e.g. hotembossing). Moreover, certain materials such as carbon based plasticsimpose less scattering of X-rays, thereby facilitating collection ofdiffraction data directly from a chip.

An additional possibility for the harvesting of crystals is to have amethod of off-loading from chip. Off-loading could be performed oncecrystals have formed, or alternatively, prior to incubation. Theseoff-loaded crystals could then be used to seed macroscopic reactions, orbe extracted and mounted in a cryo-loop. If a method for the addition ofcryogen was also developed, the crystals could be flash frozen andmounted directly into the x-ray beam.

4. Temporal Control Over Equilibration

The growth and quality of crystals is determined not only bythermodynamic conditions explored during the equilibration, but also bythe rate at which equilibration takes place. It is therefore potentiallyvaluable to control the dynamics of equilibration.

In conventional crystallization methods, course control only over thedynamics of equilibration may be available through manipulation ofinitial conditions. For macroscopic free interface diffusion, oncediffusion begins, the experimenter has no control over the subsequentequilibration rate. For hanging drop experiments, the equilibration ratemay be changed by modifying the size of the initial drop, the total sizeof the reservoir, or the temperature of incubation. In microbatchexperiments, the rate at which the sample is concentrated may be variedby manipulating the size of the drop, and the identity and amount of thesurrounding oil. Since the equilibration rates depend in a complicatedmanner on these parameters, the dynamics of equilibration may only bechanged in a coarsely manner. Moreover, once the experiment has begun,no further control over the equilibration dynamics is available.

By contrast, the fluidic interface in a gated μ-Fib experiment may becontrolled by manipulation of the dimensions of the reaction chambersand of the connecting channels. To good approximation, the time requiredfor equilibration varies as the required diffusion length. Theequilibration rate also depends on the cross-sectional area of theconnecting channels. The required time for equilibration may thereforebe controlled by changing both the length, and the cross-sectional areaof the connecting channels.

FIG. 38A shows three sets of pairs of compound chambers 9800, 9802, and9804, each pair connected by microchannels 9806 of a different lengthΔx. FIG. 38B plots equilibration time versus equilibration distance.FIG. 38B shows that the required time for equilibration of the chambersof FIG. 38A varies as the length of the connecting channels.

FIG. 39 shows four compound chambers 9900, 9902, 9904, and 9906, eachhaving different arrangements of connecting microchannel(s) 9908.Microchannels 9908 have the same length, but differ in cross-sectionalarea and/or number of connecting channels. The rate of equilibration maythus be increased/decreased by decreasing/increasing the cross-sectionalarea, for example by decreasing/increasing the number of connectingchannels or the dimensions of those channels.

Varying the equilibration rate by changing the geometry of connectingchannels may be used on a single device to explore the effect ofequilibration dynamics on crystal growth. FIGS. 37A-D show an embodimentin which a gradient of concentrations, initially established by thepartial diffusive equilibration of two solutions from a micro-freeinterface, can be maintained by the actuation of containment valves.

FIG. 37A shows flow channel 10000 that is overlapped at intervals by aforked control channel 10002 to define a plurality of chambers (A-G)positioned on either side of a separately-actuated interface valve10004. FIG. 37B plots solvent concentration at an initial time, wheninterface valve 10004 is actuated and a first half 10000 a of the flowchannel has been mixed with a first solution, and a second half 10000 bof the flow channel has been primed with a second solution. FIG. 37Cplots solvent concentration at a subsequent time T₁, when controlchannel 10002 is actuated to define the seven chambers (A-G), whichcapture the concentration gradient at that particular point in time.FIG. 37D plots relative concentration of the chambers (A-G) at time T₁.

In the embodiment shown in FIG. 37A, actuation of the forked controlchannel simultaneously creates the plurality of chambers A-G. However,this is not required, and in alternative embodiments of the presentinvention multiple control channels could be utilized to allowindependent creation of chambers A-G at different time intervals,thereby allow additional diffusion to occur after an initial set ofchambers are created immediately adjacent to the free interface.

An embodiment of a method of capturing a concentration gradient betweentwo fluids comprises providing a first fluid on a first side of anelastomer membrane present within a microfluidic flow channel, andproviding a second fluid on a second side of the elastomer membrane. Theelastomer membrane is displaced from the microfluidic flow channel todefine a microfluidic free interface between the first fluid and thesecond fluid. The first fluid and the second fluid are allowed todiffuse across the microfluidic free interface. A group of elastomervalves positioned along the flow channel at increasing distances fromthe microfluidic free interface are actuated to define a succession ofchambers whose relative concentration of the first fluid and the secondfluid reflects a time of diffusion across the microfluidic freeinterface.

5. Target Materials

Typical targets for crystallization are diverse. A target forcrystallization may include but is not limited to: 1) biologicalmacromolecules (cytosolic proteins, extracellular proteins, membraneproteins, DNA, RNA, and complex combinations thereof), 2) pre- andpost-translationally modified biological molecules (including but notlimited to, phosphorylated, sulfolated, glycosylated, ubiquitinated,etc. proteins, as well as halogenated, abasic, alkylated, etc. nucleicacids); 3) deliberately derivatized macromolecules, such as heavy-atomlabeled DNAs, RNAs, and proteins (and complexes thereof),selenomethionine-labeled proteins and nucleic acids (and complexesthereof), halogenated DNAs, RNAs, and proteins (and complexes thereof),4) whole viruses or large cellular particles (such as the ribosome,replisome, spliceosome, tubulin filaments, actin filaments, chromosomes,etc.), 5) small-molecule compounds such as drugs, lead compounds,ligands, salts, and organic or metallo-organic compounds, and 6)small-molecule/biological macromolecule complexes (e.g., drug/proteincomplexes, enzyme/substrate complexes, enzyme/product complexes,enzyme/regulator complexes, enzyme/inhibitor complexes, and combinationsthereof). Such targets are the focus of study for a wide range ofscientific disciplines encompassing biology, biochemistry, materialsciences, pharmaceutics, chemistry, and physics.

A nonexclusive listing of possible protein modifications is as follows:5′ dephospho; Desmosine (from Lysine); decomposed carboxymethylatedMethionine; Ornithine (from Arginine); Lysinoalanine (from Cysteine);Lanthionine (from Cysteine); Dehydroalanine (from Cysteine); Homoserineformed from Met by CNBr treatment; Dehydration (—H2O); S-gamma-Glutamyl(crosslinked to Cysteine); O-gamma-Glutamyl- (Crosslink to Serine);Serine to Dehydroalanine; Alaminohistidine (Serine crosslinked to thetaor pi carbon of Histidine); Pyroglutamic Acid formed from Gln;N-pyrrolidone carboxyl (N terminus); N alpha -(gamma-Glutamyl)-lysine;N-(beta-Aspartyl)-Lysine (Crosslink); 3,3′,5,5′-TerTyr (Crosslink);Disulphide bond formation (Cystine); S-(2-Histidyl)- (Crosslinked toCysteine); S-(3-Tyr) (Crosslinked to Cysteine); 3,3′-BiTyr (Crosslink);IsodiTyr (Crosslink); Allysine (from Lysine); Amide formation (Cterminus); Deamidation of Asparagine and Glutamine to Aspartate andGlutamate; Citruline (from Arginine); Syndesine (from Lysine);Methylation (N terminus, N epsilon of Lysine, O of Serine, Threonine orC terminus, N of Asparagine); delta-Hydroxy-allysine (from Lysine);Hydroxylation (of delta C of Lysine, beta C of Tryptophan, C3 or C4 ofProline, beta C of Aspartate); Oxidation of Methionine (to Sulphoxide);Sulfenic Acid (from Cysteine); Pyruvoyl-(Serine);3,4-Dihydroxy-Phenylalanine (from Tyrosine) (DOPA); Sodium; Ethyl; N,Ndimethylation (of Arginine or Lysine); 2,4-BisTrp-6,7-dione (fromTryptophan); Formylation (CHO); 6,7 Dione (from Tryptophan);3,4,6-Trihydroxy-Phenylalanine (from Tyrosine) (TOPA);3,4-Dihydroxylation (of Proline); Oxidation of Methionine (to Sulphone);3-Chlorination (of Tyrosine with 35Cl); 3-Chlorination (of Tyrosine with37Cl); Potassium; Carbamylation; Acetylation (N terminus, N epsilon ofLysine, O of Serine) (Ac); N-Trimethylation (of Lysine); gammaCarboxylation of Glutamate or beta Carboxylation of Aspartate; disodium;Nitro (NO2); t-butyl ester(OtBu) and t-butyl (tBu); Glycyl (-G-,-Gly-);Carboxymethyl (on Cystine); sodium+potassium; Selenocysteine (fromSerine); 3,5-Dichlorination (of Tyrosine with 35Cl); Dehydroalanine(Dha); 3,5-Dichlorination (of Tyrosine with mixture of 35Cl and 37Cl));Pyruvate; Acrylamidyl or Acrylamide adduct; Sarcosyl; Alanyl (-A-,-Ala-); Acetamidomethyl (Acm); 3,5-Dichlorination (of Tyrosine with37Cl); S-(sn-1-Glyceryl) (on Cysteine); Glycerol Ester (on Glutamic acidside chain); Glycine (G, Gly); Beta mercaptoethanol adduct; Phenyl ester(OPh) (on acidic); 3-Bromination (of Tyrosine with 79Br);Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, Nepsilon of Lysine); 3-Bromination (of Tyrosine with 81Br); Sulphonation(SO3H) (of PMC group); Sulphation (of O of Tyrosine); Cyclohexyl ester(OcHex); Homoseryl lactone; Dehydroamino butyric acid (Dhb); GammaAminobutyryl; 2-Aminobutyric acid (Abu); 2-Aminoisobutyric acid (Aib);Diaminopropionyl; t-butyloxymethyl (Bum); N-(4-NH2-2-OH-butyl)- (ofLysine) (Hypusine); Seryl (-S-, -Ser-); t-butylsulfenyl (StBu); Alanine(A, Ala); Sarcosine (Sar); Anisyl; Benzyl (Bzl) and benzyl ester (OBzl);1,2-ethanedithiol (EDT); Dehydroprolyl; Trifluoroacetyl (TFA);N-hydroxysuccinimide (ONSu, OSu); Prolyl (-P-, -Pro-); Valyl (-V-,-Val-); Isovalyl (-I-,-Iva-); t-Butyloxycarbonyl (tBoc); Threoyl (-T-,-Thr-); Homoseryl (-Hse-); Cystyl (-C-, -Cys-); Benzoyl (Bz);4-Methylbenzyl (Meb); Serine (S, Ser); HMP (hydroxymethylphenyl) linker;Thioanisyl; Thiocresyl; Diphthamide (from Histidine); Pyroglutamyl;2-Piperidinecarboxylic acid (Pip); Hydroxyprolyl (-Hyp-); Norleucyl(-Nle-); Isoleucyl (-I-, -Ile-); Leucyl (-L-, -Leu-); Ornithyl (-Orn-);Asparagyl (-N-, -Asn-); t-amyloxycarbonyl (Aoc); Proline (P, Pro);Aspartyl (-D-, -Asp-); Succinyl; Valine (V, Val); Hydroxybenzotriazoleester (HOBt); Dimethylbenzyl (diMeBzl); Threonine (T, Thr);Cysteinylation; Benzyloxymethyl (Bom); p-methoxybenzyl (Mob, Mbzl);4-Nitrophenyl, p-Nitrophenyl (ONp); Cysteine (C, Cys); Chlorobenzyl(ClBzl); Iodination (of Histidine[C4] or Tyrosine[C3]); Glutamyl (-Q-,-Gln-); N-methyl Lysyl; Lysyl (-K-, -Lys-); O-Methyl Aspartamyl;Glutamyl (-E-, -Glu-); N alpha -(gamma-Glutamyl)-Glu; Norleucine (Nle);Hydroxy Aspartamyl; Hydroxyproline (Hyp); bb-dimethyl Cystenyl;Isoleucine (I, Ile); Leucine (L, Leu); Methionyl (-M-, -Met-);Asparagine (N, Asn); Pentoses (Ara, Rib, Xyl); Aspartic Acid (D, Asp);Dmob (Dimethoxybenzyl); Benzyloxycarbonyl (Z); Adamantyl (Ada);p-Nitrobenzyl ester (ONb); Histidyl (-H-, -His-); N-methyl Glutamyl;O-methyl Glutamyl; Hydroxy Lysyl (-Hyl-); Methyl Methionyl; Glutamine(Q, Gln); Aminoethyl Cystenyl; Pentosyl; Deoxyhexoses (Fuc, Rha); Lysine(K, Lys); Aminoethyl cystenyl (-AECys-); 4-Glycosyloxy- (pentosyl, C5)(of Proline); Methionyl Sulfoxide; Glutamic Acid (E, Glu); Phenylalanyl-(-F-, -Phe-); Pyridyl Alanyl; Fluorophenylalanyl; 2-Nitrobenzoyl (NBz);Methionine (M, Met); 3-methyl Histidyl; 2-Nitrophenylsulphenyl (Nps);4-Toluenesulphonyl (Tosyl, Tos); 3-nitro-2-pyridinesulfenyl (Npys);Histidine (H, His); 3,5-Dibromination (of Tyrosine with 79Br); Arginyl(-R-, -Arg-); Citrulline; 3,5-Dibromination (of Tyrosine with mixture of79Br and 81Br); Dichlorobenzyl (Dcb); 3,5-Dibromination (of Tyrosinewith 81Br); Carboxyamidomethyl Cystenyl; Carboxymethyl Cystenyl;Methylphenylalanyl; Hexosamines (GalN, GlcN); Carboxymethyl cysteine(Cmc); N-Glucosyl (N terminus or N epsilon of Lysine) (Aminoketose);O-Glycosyl- (to Serine or Threonine); Hexoses (Fru, Gal, Glc, Man);Inositol; MethionylSulphone; Tyrosinyl (-Y-, -Tyr-); Phenylalanine (F,Phe); 2,4-dinitrophenyl (Dnp); Pentafluorophenyl (Pfp); Diphenylmethyl(Dpm); Phospho Seryl; 2-Chlorobenzyloxycarbonyl (ClZ); Napthyl acetyl;Isopropyl Lysyl; N-methyl Arginyl; Ethaneditohiol/TFA cyclic adduct;Carboxy Glutamyl (Gla); Acetamidomethyl Cystenyl; Acrylamidyl Cystenyl;Arginine (R. Arg); N-Glucuronyl (N terminus); delta-Glycosyloxy- (ofLysine) or beta-Glycosyloxy- (of Phenylalanine or Tyrosine);4-Glycosyloxy- (hexosyl, C6) (of Proline); Benzyl Seryl; N-methylTyrosinyl; p-Nitrobenzyloxycarbonyl (4Nz); 2,4,5-Trichlorophenyl;2,4,6-trimethyloxybenzyl (Tmob); Xanthyl (Xan); Phospho Threonyl;Tyrosine (Y, Tyr); Chlorophenylalanyl; Mesitylene-2-sulfonyl (Mts);Carboxymethyl Lysyl; Tryptophanyl (-W-, -Trp-); N-Lipoyl- (on Lysine);Matrix alpha cyano MH+; Benzyl Threonyl; Benzyl Cystenyl; NapthylAlanyl; Succinyl Aspartamyl; Succinimidophenyl carb.; HMP(hydroxymethylphenyl)/TFA adduct; N-acetylhexosamines (GalNAc, GlcNAc);Tryptophan (W, Trp); Cystine ((Cys)2); Farnesylation; S-Farnesyl-;Myristoleylation (myristoyl with one double bond); PyridylethylCystenyl; Myristoylation; 4-Methoxy-2,3,6-trimethylbenzenesulfonyl(Mtr); 2-Bromobenzyloxycarbonyl (BrZ); Formyl Tryptophanyl; BenzylGlutamyl; Anisole Adducted Glutamyl; S-cystenyl Cystenyl;9-Flourenylmethyloxycarbonyl (Fmoc); Lipoic acid (amide bond to lysine);Biotinylation (amide bond to lysine); Dimethoxybenzhydryl (Mbh);N-Pyridoxyl (on Lysine); Pyridoxal phosphate (Schiff Base formed tolysine); Nicotinyl Lysyl; Dansyl (Dns);2-(p-biphenyl)isopropyl-oxycarbonyl (Bpoc); Palmitoylation;“Triphenylmethyl (Trityl, Trt)”; Tyrosinyl Sulphate; Phospho Tyrosinyl;Pbf (pentamethyldihydrobenzofuransulfonyl); 3,5-Diiodination (ofTyrosine); 3,5-di-I”; N alpha -(gamma-Glutamyl)-Glu2;O-GlcNAc-1-phosphorylation (of Serine);“2,2,5,7,8-Pentamethylchroman-6-sulphonyl (Pmc)”; Stearoylation;Geranylgeranylation; S-Geranylgeranyl; 5′phos dCytidinyl; iodoTyrosinyl; Aldohexosyl Lysyl; Sialyl; N-acetylneuraminic acid (Sialicacid, NeuAc, NANA, SA); 5′phos dThymidinyl; 5′phos Cytidinyl;Glutathionation; O-Uridinylylation (of Tyrosine); 5′phos Uridinyl;S-farnesyl Cystenyl; N-glycolneuraminic acid (NeuGc); 5′phos dAdenosyl;O-pantetheinephosphorylation (of Serine); SucPhencarb Lysyl; 5′phosdGuanosyl; 5′phos Adenosinyl; O-5′-Adenosylation (of Tyrosine);4′-Phosphopantetheine; GL2; S-palmityl Cystenyl; 5′phos Guanosyl;Biotinyl Lysyl; Hex-HexNAc; N alpha -(gamma-Glutamyl)- Glu3; DioctylPhthalate; PMC Lysyl; Aedans Cystenyl; Dioctyl Phthalate Sodium Adduct;di-iodo Tyrosinyl; PMC Arginyl; S-Coenzyme A; AMP Lysyl;3,5,3′-Triiodothyronine (from Tyrosine); S-(sn-1-Dipalmitoyl-glyceryl)-(on Cysteine); S-(ADP-ribosyl)- (on Cysteine); N-(ADP-ribosyl)- (onArginine); O-ADP-ribosylation (on Glutamate or C terminus);ADP-rybosylation (from NAD); S-Phycocyanobilin (on Cysteine); S-Heme (onCysteine); N theta -(ADP-ribosyl) diphthamide (of Histidine);NeuAc-Hex-HexNAc; MGDG; O-8 alpha-Flavin [FAD])- (of Tyrosine);S-(6-Flavin [FAD])- (on Cysteine); N theta and N pi-(8alpha-Flavin) (onHistidine); (Hex)3-HexNAc-HexNAc; (Hex)3-HexNAc-(dHex)HexNAc.

A nonexclusive listing of possible nucleic acid modifications, such asbase-specific, sugar-specific, or phospho-specific is as follows:halogenation (F, Cl, Br, I); Abasic sites; Alkylation; Crosslinkableadducts such as thiols or azides; Thiolation; Deamidation;Fluorescent-group labeling, and glycosylation.

A nonexclusive listing of possible heavy atom derivatizing agents is asfollows: potassium hexachloroiridate (III); Potassium hexachloroiridate(IV); Sodium hexachloroiridate (IV); Sodium hexachloroiridate (III);Potassium hexanitritoiridate (III); Ammonium hexachloroiridate (III);Iridium (III) chloride; Potassium hexanitratoiridate (III); Iridium(III) bromide; Barium (II) chloride; Barium (II) acetate; Cadmium (II)nitrate; Cadmium (II) iodide; Lead (II) nitrate; Lead (II) acetate;Trimethyl lead (IV) chloride; Trimethyl lead (IV) acetate; Ammoniumhexachloro plumbate (IV); Lead (II) chloride; Sodium hexachlororhodiate(III); Strontium (II) acetate; Disodium thiomalonato aurate (I);Potassium dicyano aurate (I); Sodium dicyano aurate (I); Sodiumthiosulphato aurate (III); Potassium tetracyano aurate (III); Potassiumtetrachloro aurate (III); Hydrogen tetrachloro aurate (III); Sodiumtetrachloro aurate (III); Potassium tetraiodo aurate (III); Potassiumtetrabromo aurate (III); (acetato-o)methylmercury; Methyl (nitrato-o)mercury; Chloromethylmercury; Iodomethylmercury; Chloroethylmercury;Methyl mercury cation; Triethyl (m3-phosphato(3-)-0,0′,0″)tri mercuryeth; [3-[(aminocarbonyl)amino]-2-methoxypropyl]chlorome; 1,4diacetoxymercury 2-3 dimethoxy butane; Meroxyl mercuhydrin; Tetrakis(acetoxy mercuri)-methane; 1,4-bis(chloromercuri)-2,3-butanediol; Ethyldiacetoxymercurichloro acetate (dame); Mercuric (II) oxide; Methylmercuri-2-mercaptoethanol; 3,6 bis(mercurimethyl dioxane acetate); Ethylmercury cation; Billman's dimercurial; Para chloromercury phenyl acetate(pcma); Mercury phenyl glyoxal (mpg); Thiomersal, ethyl mercurythiosalicylate [emts]; 4-chloromercuribenesulphonic acid; 2,6dichloromercuri-4-nitrophenol (dcmnp);[3-[[2(carboxymethoxy)benzoyl]mino-2 methoxy prop; Parachloromercurybenzoate (pcmb), 4-chloromercury; (acetato-o)phenyl mercury; Phenylmercuri benzoate (pmb); Para hydroxy mercuri benzoate (phmb); Mercuricimidosuccinate/mercury succinimide; 3-hydroxymercurybenzaldehyde;2-acetoxy mercuri sulhpanilamide;3-acetoxymercuri-4-aminobenzenesulphonamide; Methyl mercuri thioclycolicacid (mmtga); 2-hydroxymercuri-tolulen-4-sulphonic acid (hmts);Acetamino phenyl mercury acetate (apma);[3-[(aminocarbonyl)amino]-3-methoxypropyl 2-chloro; Para-hydroxymercuribenzene sulphonate (phmbs); Ortho-chloromercuri phenol (ocmp);Diacetoxymercury dipopylene dioxide (dmdx); Para-acetoxymercuri aniline(pama); (4-aminophenyl) chloromercury; Aniline mercury cation;3-hydroxy-mercuri-s sulphosalicylic acid (msss); 3 or 5 hydroxymercurisalicylic acid (hmsa); Diphenyl mercury; 2,6 diacetoxymercurimethyl 1-4thioxane (dmmt); 2,5-b1s (chloromercury) furan; Ortho-chloromercurinitrophenol (ocmnp); 5-mercurydeoxyuridine monosulphate; Mercurysalicylate; [3-[[2-(carboxymethoxy)benzoyl]amino-2-methoxypro; 3,3bis(hydroximercuri)-3-nitratomercuri pyruvic; 3-chloro mercuri pyridine;3,5 bis acetoxymercuri methyl morpholine; Ortho-mercury phenol cation;Para-carboxymethyl mercaptomercuri benzensulphonyl; Para-mercuribenzoylglucosamine; 3-acetetoxymercuri-5-nitrosalicyladehyde (msa); Ammoniumtetrachloro mercurate (II); Potassium tetrathiocyanato mercurate (II);Sodium tetrathiocyanato mercurate (II); Potassium tetraisothiocyantomercurate (II); Potassium tetraido mercurate (II); Ammoniumtetrathiocyananato mercurate (II); Potassium tetrabromo mercurate (II);Potassium tetracyano mercurate (II); Mercury (II) bromide; Mercury (II)thiocyanate; Mercury (II) cyanide; Mercury (II) iodide; Mercuric (II)chloride; Mercury (II) acetate; Mercury (I) acetate; Dichlorodiaminomercurate (II); Beta mercury-mercapto-ethylamine hydrochloride; Mercury(II) sulphate; Mercury (II) chloroanilate; Dimercuriacetate;Chloro(2-oxoethyl) mercury; Phenol mercury nitrate; Mercurymercaptoethanol; Mercury mercaptoethylamine chloride; Mercurythioglycollic acid (sodium salt);O-hydroxymercuri-p-nitrophenol/2-hydroxymercuri-4-; Para chloromercuriphenol (pcmp); Acetylmercurithiosalicylate (amts); Iodine; Potassiumiodide (iodine); 4-iodopyrazole; O-iodobenzoylglucasamine;P-iodobenzoylglucasamine; Potassium iodide/chloramine t; Ammoniumiodide; 3-isothio-cyanato-4-iodobenzene sulphonate; Potassium iodide;3′-iodo phenyltrazine; 4′-iodo phenyltrazine; Sodium iodide/iodine;Silver nitrate; Silver ( ) trinitridosulphoxylate; Tobenamed; Samarium(III) chloride; Thulium (III) chloride; Lutetium (III) chloride;Europium (III) chloride; Terbium (III) chloride; Gadolinium (III)chloride; Erbium (III) chloride; Lanthanum (III) chloride; Samarium(III) nitrate; Samarium (III) acetate; Samarium (III) cation;Praseodymium (III) chloride; Neodymium (III) chloride; Ytterbium (III)chloride; Thulium (III) sulphate; Ytterbium (III) sulphate; Gadolinium(III) sulphate; Gadolinium (III) acetate; Dysprosium (III) chloride;Erbium (III) nitrate; Holmium (III) chloride; Penta amino ruthenium(III) chloride; Cesium nitridotiroxo osmium (viii); Potassium tetraoxoosmiate; Hexa amino osmium (III) iodide; Ammonium hexachloroosmiate(IV); Osmium (III) chloride; Potassium hexachloro osmiate (IV); Cesiumtrichloro triscarbonyl osmiate (?); Dinitritodiamine platinum (II); Cisdichlorodimethylammido platinum (II); Dichlorodiammine platinum (II);Dibromodiammine platinum (II); Dichloroethylene diamine platinum (II);Potassium dicholodinitrito platinate (II); Diethylenediamene platinum(II); Potassium dioxylato platinate (II); Dichlorobis (pyridine)platinum (II); Potassium (thimethyl dibenzyloamine) platinum (?);Potassium tetrabromoplatinate (II); Potassium tetrachloro platinate(II); Potassium tetranitrito platinum (II); Potassium tetracyanoplatinate (II); Sodium tetracyano platinate (II); Potassiumtetrathiocyanato platinate (II); Ammonium tetranitrito platinate (II);Potassium tetraisocyanato platinate (II); Ammonium tetracyano platinum(II); Ammonium tetrachloro platinate (II); Potassium dinitritodioxalatoplatinate (IV); Dichlorotetraammino platinium (IV); Dibromodinitritodiammine platinium (IV); Potassium hexanitrito platinate (IV); Potassiumhexachloro platinate (IV); Potassium hexabromo platinate (IV); Sodiumhexachloroplatinate (IV); Potassium hexaiodo platinate (IV); Potassiumhexathiocyanato platinate (IV); Tetrachloro bis(pyridine) platinum (IV);Ammonium hexachloro platinate (IV); Di-mu-iodo bis(ethylenediamine) diplatinum (II) n; Potassium hexaisothiocyanato platinate (IV); Potassiumtetraiodo platinate (II); 2,2′,2″ terpyridyl platinium (II); 2hydroxyethanethiolate (2,2′,2″ terpyeidine) pla; Potassium tetranitroplatinate (II); Trimethyl platinum (II) nitrate; Sodium tetraoxo rhenate(VII); Potassium tetraoxo rhenate (VI); Potassium tetraoxo rhenate(VII); Potassium hexachloro rhenium (IV); Rhenium (III) chloride;Ammonium hexachloro rhenate (IV); Dimethyltin (II) dichloride; Thorium(IV) nitrate; Uranium (VI) oxychloride; Uranium (VI) oxynitrate; Uranium(VI) oxyacetate; Uranium (VI) oxypyrophosphate; Potassium pentafluorooxyuranate (VI); Sodium pentafluoro oxyuranate (VI); Potassiumnanofluoro dioxyuranate (VI); Sodium triacetate oxyuranate (VI); Uranium(VI) oxyoxalate; Selenocyanate anion; Sodium tungstate; Sodium12-tungstophosphate; Thallium (I) acetate; Thallium (I) fluoride;Thallium (I) nitrate; Potassium tetrachloro palladate (II); Potassiumtetrabromo palladate (II); Potassium tetracyano palladate (II);Potassium tetraiodo palladate (II); Cobalt (II) chloride.

The PDMS material from which the chip can be formed is well suited formany of these targets, particularly biological samples. PDMS is anon-reactive and biologically inert compound that allows such moleculesto maintain their appropriate shape, fold, and activity in a solublizedstate. The matrix and system can accommodate a range of target sizes andmolecular weights, from a few hundred Daltons to the mega-Dalton regime.Biological targets, from small proteins and peptides to viruses andmacromolecular complexes, fall within this range, and are generallyanywhere from 3-10 kDa to >1-2 MDa in size.

6. Solute/Reagent Types

During crystallization screening, a large number of chemical compoundsmay be employed. These compounds include salts, small and largemolecular weight organic compounds, buffers, ligands, small-moleculeagents, detergents, peptides, crosslinking agents, and derivatizingagents. Together, these chemicals can be used to vary the ionicstrength, pH, solute concentration, and target concentration in thedrop, and can even be used to modify the target. The desiredconcentration of these chemicals to achieve crystallization is variable,and can range from nanomolar to molar concentrations. A typicalcrystallization mix contains set of fixed, but empirically-determined,types and concentrations of ‘precipitants’, buffers, salts, and otherchemical additives (e.g., metal ions, salts, small molecular chemicaladditives, cryo protectants, etc.). Water is a key solvent in manycrystallization trials of biological targets, as many of these moleculesmay require hydration to stay active and folded.

As described above in connection with the pressurized out-gas priming(POP) technique, the permeability of PDMS to gases, and thecompatibility of solvents with PDMS may be a significant factor indeciding upon precipitating agents to be used.

‘Precipitating’ agents act to push targets from a soluble to insolublestate, and may work by volume exclusion, changing the dielectricconstant of the solvent, charge shielding, and molecular crowding.Precipitating agents compatible with the PDMS material of certainembodiments of the chip include, but are not limited to, non-volatilesalts, high molecular weight polymers, polar solvents, aqueoussolutions, high molecular weight alcohols, divalent metals.

Precipitating compounds, which include large and small molecular weightorganics, as well as certain salts are used from under 1% to upwards of40% concentration, or from <0.5M to greater than 4M concentration. Wateritself can act in a precipitating manner for samples that require acertain level of ionic strength to stay soluble. Many precipitants mayalso be mixed with one another to increase the chemical diversity of thecrystallization screen. The microfluidics devices described in thisdocument are readily compatible with a broad range of such compounds.Moreover, many precipitating agents (such as long- and short-chainorganics) are quite viscous at high concentrations, presenting a problemfor most fluid handling devices, such as pipettes or robotic systems.The pump and valve action of microfluidics devices in accordance withembodiments of the present invention enable handling of viscous agents.

An investigation of solvent/precipitating agent compatibility withparticular elastomer materials may be conducted to identify optimumcrystallizing agents, which may be employed develop crystallizationscreening reactions tailored for the chip that are more effective thanstandard screens.

A nonexclusive list of salts which may be used as precipitants is asfollows: Tartrate (Li, Na, K, Na/K, NH4); Phosphate (Li, Na, K, Na/K,NH4); Acetate (Li, Na, K, Na/K, Mg, Ca, Zn, NH4); Formate (Li, Na, K,Na/K, Mg, NH4); Citrate (Li, Na, K, Na/K, NH4); Chloride (Li, Na, K,Na/K, Mg, Ca, Zn, Mn, Cs, Rb, NH4); Sulfate (Li, Na, K, Na/K, NH4);Malate (Li, Na, K, Na/K, NH4); Glutamate (Li, Na, K, Na/K, NH4.

A nonexclusive list of organic materials which may be used asprecipitants is as follows: PEG 400; PEG 1000; PEG 1500; PEG 2k; PEG3350; PEG 4k; PEG 6k; PEG 8k; PEG 10k; PEG 20k; PEG-MME 550; PEG-MME750; PEG-MME 2k; PEG-MME 5k; PEG-DME 2k; Dioxane; Methanol; Ethanol;2-Butanol; n-Butanol; t-Butanol; Jeffamine M-600; Isopropanol;2-methyl-2,4-pentanediol; 1,6 hexanediol.

Solution pH can be varied by the inclusion of buffering agents; typicalpH ranges for biological materials lie anywhere between values of3.5-10.5 and the concentration of buffer, generally lies between 0.01and 0.25 M. The microfluidics devices described in this document arereadily compatible with a broad range of pH values, particularly thosesuited to biological targets.

A nonexclusive list of possible buffers is as follows: Na-Acetate;HEPES; Na-Cacodylate; Na-Citrate; Na-Succinate; Na-K-Phosphate; TRIS;TRIS-Maleate; Imidazole-Maleate; BisTrisPropane; CAPSO, CHAPS, MES, andimidizole.

Additives are small molecules that affect the solubility and/or activitybehavior of the target. Such compounds can speed crystallizationscreening or produce alternate crystal forms of the target. Additivescan take nearly any conceivable form of chemical, but are typically monoand polyvalent salts (inorganic or organic), enzyme ligands (substrates,products, allosteric effectors), chemical crosslinking agents,detergents and/or lipids, heavy metals, organo-metallic compounds, traceamounts of precipitating agents, and small molecular weight organics.

The following is a nonexclusive list of possible additives: 2-Butanol;DMSO; Hexanediol; Ethanol; Methanol; Isopropanol; sodium fluoride;potassium fluoride; ammonium fluoride; lithium chloride anhydrous;magnesium chloride hexahydrate; sodium chloride; Calcium chloridedihydrate; potassium chloride; ammonium chloride; sodium iodide;potassium iodide; ammonium iodide; sodium thiocyanate; potassiumthiocyanate; lithium nitrate; magnesium nitrate hexahydrate; sodiumnitrate; potassium nitrate; ammonium nitrate; magnesium formate; sodiumformate; potassium formate; ammonium formate; lithium acetate dihydrate;magnesium acetate tetrahydrate; zinc acetate dihydrate; sodium acetatetrihydrate; calcium acetate hydrate; potassium acetate; ammoniumacetate; lithium sulfate monohydrate; magnesium sulfate heptahydrate;sodium sulfate decahydrate; potassium sulfate; ammonium sulfate;di-sodium tartate dihydrate; potassium sodium tartrate tetrahydrate;di-ammonium tartrate; sodium dihydrogen phosphate monohydrate; di-sodiumhydrogen phosphate dihydrate; potassium dihydrogen phosphate;di-potassium hydrogen phosphate; ammonium dihydrogen phosphate;di-ammonium hydrogen phosphate; tri-lithium citrate tetrahydrate;tri-sodium citrate dihydrate; tri-potassium citrate monohydrate;di-ammonium hydrogen citrate; barium chloride; cadmium chloridedihydrate; cobaltous chloride dihydrate; cupric chloride dihydrate;strontium chloride hexahydrate; yttrium chloride hexahydrate; ethyleneglycol; Glycerol anhydrous; 1,6 hexanediol; MPD; polyethylene glycol400; trimethylamine HCl; guanidine HCl; urea; 1,2,3-heptanetriol;benzamidine HCl; dioxane; ethanol; iso-propanol; methanol; sodiumiodide; L-cysteine; EDTA sodium salt; NAD; ATP disodium salt;D(+)-glucose monohydrate; D(+)-sucrose; xylitol; spermidine; sperminetetra-HCl; 6-aminocaproic acid; 1,5-diaminopentane di-HCl;1,6-diaminohexane; 1,8-diaminooctane; glycine; glycyl-glycyl-glycine;hexaminecobalt trichloride; taurine; betaine monohydrate;polyvinylpyrrolidone K15; non-detergent sulfo-betaine 195; non-detergentsulfo-betaine 201; phenol; DMSO; dextran sulfate sodium salt; jeffamineM-600; 2,5 Hexanediol; (+/−)-1,3 butanediol; polypropylene glycol P400;1,4 butanediol; tert-butanol; 1,3 propanediol; acetonitrile; gammabutyrolactone; propanol; ethyl acetate; acetone; dichloromethane;n-butanol; 2,2,2 trifluoroethanol; DTT; TCEP; nonaethylene glycolmonododecyl ether, nonaethylene glycol monolauryl ether; polyoxyethylene(9) ether; octaethylene glycol monododecyl ether, octaethylene glycolmonolauryl ether; polyoxyethylene (8) lauryl ether;Dodecyl-β-D-maltopyranoside; Lauric acid sucrose ester;Cyclohexyl-pentyl-β-D-maltoside; Nonaethylene glycol octylphenol ether;Cetyltrimethylammonium bromide;N,N-bis(3-D-gluconamidopropyl)-deoxycholamine;Decyl-β-D-maltopyranoside; Lauryldimethylamine oxide;Cyclohexyl-pentyl-β-D-maltoside; n-Dodecylsulfobetaine,3-(Dodecyldimethylammonio)propane-1-sulfonate;Nonyl-β-D-glucopyranoside; Octyl-β-D-thioglucopyranoside, OSG;N,N-Dimethyldecylamine-B-oxide;Methyl-6-O-(N-heptylcarbamoyl)-a-D-glucopyranoside; Sucrosemonocaproylate; n-Octanoyl-β-D-fructofuranosyl-a-D-glucopyranoside;Heptyl-β-D-thioglucopyranoside; Octyl-β-D-glucopyranoside, OG;Cyclohexyl-propyl-β-D-maltoside;Cyclohexylbutanoyl-N-hydroxyethylglucamide; n-decylsulfobetaine,3-(Decyldimethylammonio)propane-1-sulfonate; Octanoyl-N-methylglucamide,OMEGA; Hexyl-β-D-glucopyranoside; Brij 35; Brij 58; Triton X-114; TritonX-305; Triton X-405; Tween 20; Tween 80; polyoxyethylene(6)decyl ether;polyoxyethylene(9)decyl ether; polyoxyethylene(10)dodecyl ether;polyoxyethylene(8)tridecyl ether; Isopropyl-β-D-thiogalactoside;Decanoyl-N-hydroxyethylglucamide; Pentaethylene glycol monooctyl ether;3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate;3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propane sulfonate;Cyclohexylpentanoyl-N-hydroxyethylglucamide;Nonanoyl-N-hydroxyethyglucamide;Cyclohexylpropanol-N-hydroxyethylglucamide;Octanoyl-N-hydroxyethylglucamide;Cyclohexylethanoyl-N-hydroxyethylglucamide; Benzyldimethyldodecylammonium bromide; n-Hexadecyl-β-D-maltopyranoside;n-Tetradecyl-β-D-maltopyranoside; n-Tridecyl-β-D-maltopyranoside;Dodecylpoly(ethyleneglycoether)n;n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;n-Undecyl-β-D-maltopyranoside; n-Decyl-β-D-thiomaltopyranoside;n-dodecylphosphocholine; a-D-glucopyranoside, β-D-fructofuranosylmonodecanoate, sucrose mono-caprate; 1-s-Nonyl-β-D-thioglucopyranoside;n-Nonyl-β-D-thiomaltoyranoside; N-Dodecyl-N,N-(dimethlammonio)butyrate;n-Nonyl-β-D-maltopyranoside; Cyclohexyl-butyl-β-D-maltoside;n-Octyl-β-D-thiomaltopyranoside; n-Decylphosphocholine;n-Nonylphosphocho line; Nonanoyl-N-methylglucamide;1-s-Heptyl-β-D-thioglucopyranoside; n-Octylphosphocholine;Cyclohexyl-ethyl-β-D-maltoside;n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;Cyclohexyl-methyl-β-D-maltoside.

Cryosolvents are agents that stabilize a target crystal to flash-coolingin a cryogen such as liquid nitrogen, liquid propane, liquid ethane, orgaseous nitrogen or helium (all at approximately 100-120° K) such thatcrystal becomes embedded in a vitreous glass rather than ice. Any numberof salts or small molecular weight organic compounds can be used as acryoprotectant, and typical ones include but are not limited to: MPD,PEG-400 (as well as both PEG derivatives and higher molecular-weight PEGcompounds), glycerol, sugars (xylitol, sorbitol, erythritol, sucrose,glucose, etc.), ethylene glycol, alcohols (both short- and long chain,both volatile and nonvolatile), LiOAc, LiCl, LiCHO₂, LiNO₃, Li2SO₄,Mg(OAc)₂, NaCl, NaCHO₂, NaNO₃, etc. Again, materials from whichmicrofluidics devices in accordance with the present invention arefabricated may be compatible with a range of such compounds.

Many of these chemicals can be obtained in predefined screening kitsfrom a variety of vendors, including but not limited to Hampton Researchof Laguna Niguel, Calif., Emerald Biostructures of Bainbridge Island,Wash., and Jena BioScience of Jena, Germany, that allow the researcherto perform both ‘sparse matrix’ and ‘grid’ screening experiments. Sparsematrix screens attempt to randomly sample as much of precipitant,buffer, and additive chemical space as possible with as few conditionsas possible. Grid screens typically consist of systematic variations oftwo or three parameters against one another (e.g., precipitantconcentration vs. pH). Both types of screens have been employed withsuccess in crystallization trials, and the majority of chemicals andchemical combinations used in these screens are compatible with the chipdesign and matrices in accordance with embodiments of the presentinvention.

Moreover, current and future designs of microfluidic devices may enableflexibly combinatorial screening of an array of different chemicalsagainst a particular target or set of targets, a process that isdifficult with either robotic or hand screening. This latter aspect isparticularly important for optimizing initial successes generated byfirst-pass screens.

7. Additional Screening Variables for Crystallization

In addition to chemical variability, a host of other parameters can bevaried during crystallization screening. Such parameters include but arenot limited to: 1) volume of crystallization trial, 2) ratio of targetsolution to crystallization solution, 3) target concentration, 4)co-crystallization of the target with a secondary small ormacromolecule, 5) hydration, 6) incubation time, 7) temperature, 8)pressure, 9) contact surfaces, 10) modifications to target molecules,and 11) gravity.

Volumes of crystallization trials can be of any conceivable value, fromthe picoliter to milliliter range. Typical values may include but arenot limited to: 0.1, 0.2, 0.25, 0.4, 0.5, 0.75, 1, 2, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200,225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 900,1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2250, 2500, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, and 10000nL. The microfluidics devices previously described can access thesevalues.

In particular, access to the low volume range for crystallization trials(<100 nL) is a distinct advantage of embodiments of the microfluidicschips in accordance with embodiments of the present invention, as suchsmall-volume crystallization chambers can be readily designed andfabricated, minimizing the need the need for large quantities ofprecious target molecules. The low consumption of target material ofembodiments in accordance with the present invention is particularlyuseful in attempting to crystallize scarce biological samples such asmembrane proteins, protein/protein and protein/nucleic acid complexes,and small-molecule drug screening of lead libraries for binding totargets of interest.

The ratios of a target solution to crystallization mix can alsoconstitute an important variable in crystallization screening andoptimization. These rations can be of any conceivable value, but aretypically in the range of 1:100 to 100:1 target:crystallization-solution. Typical target: crystallization-solution orcrystallization-solution: target ratios may include but are not limitedto: 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:25, 1:20, 1:15,1:10, 1:9, 1:8, 1:7.5, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1, 2:3,3:4, 3:5, 4:5, 5:6, 5:7, 5:9, 6:7, 7:8, 8:9, and 9:10. As previouslydescribed, microfluidics devices in accordance with embodiments of thepresent invention can be designed to access multiple ratiossimultaneously on a single chip.

Target concentration, like crystallization chemical concentration, canlie in a range of values and is an important variable in crystallizationscreening. Typical ranges of concentrations can be anywhere from <0.5mg/ml to >100 mg/ml, with most commonly used values between 5-30 mg/ml.The microfluidics devices in accordance with embodiments of the presentinvention are readily compatible with this range of values.

Co-crystallization generally describes the crystallization of a targetwith a secondary factor that is a natural or non-natural bindingpartner. Such secondary factors can be small, on the order of about10-1000 Da, or may be large macromolecules. Co-crystallization moleculescan include but are not limited to small-molecule enzyme ligands(substrates, products, allosteric effectors, etc.), small-molecule drugleads, single-stranded or double-stranded DNAs or RNAs, complementproteins (such as a partner or target protein or subunit), monoclonalantibodies, and fusion-proteins (e.g., maltose binding proteins,glutathione S-transferase, protein-G, or other tags that can aidexpression, solubility, and target behavior). As many of these compoundsare either biological or of a reasonable molecular weight,co-crystallization molecules can be routinely included with screens inthe microfluidics chips. Indeed, because many of these reagents areexpensive and/or of limited quantity, the small-volumes afforded by themicrofluidics chips in accordance with embodiment of the presentinvention make them ideally suited for co-crystallization screening.

Hydration of targets can be an important consideration. In particular,water is by far the dominant solvent for biological targets and samples.The microfluidics devices described in this document are relativelyhydrophobic, and are compatible with water-based solutions.

The length of time for crystallization experiments can range fromminutes or hours to weeks or months. Most experiments on biologicalsystems typically show results within 24 hours to 2 weeks. This regimeof incubation time can be accommodated by the microfluidics devices inaccordance with embodiments of the present invention.

The temperature of a crystallization experiment can have a great impacton success or failure rates. This is particularly true for biologicalsamples, where temperatures of crystallization experiments can rangefrom 0-42° C. Some of the most common crystallization temperatures are:0, 1, 2, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 37, and 42.Microfluidics devices in accordance with embodiments of the presentinvention can be stored at the temperatures listed, or alternatively maybe placed into thermal contact with small temperature control structuressuch as resistive heaters or Peltier cooling structures.

In addition, the small footprint and rapid setup time of embodiments inaccordance with the present invention allow faster equilibration todesired target temperatures and storage in smaller incubators at a rangeof temperatures. Moreover, as the microfluidics systems in accordancewith embodiments of the present invention do not place thecrystallization experiment in contact with the vapor phase, condensationof water from the vapor phase into the drop as temperatures change, aproblem associated with conventional macroscopic vapor-diffusiontechniques, is avoided. This feature represents an advance over manyconventional manual or robotic systems, where either the system must bemaintained at the desired temperature, or the experiment must remain atroom temperature for a period before being transferred to a newtemperature.

Variation in pressure is an as yet understudied crystallizationparameter, in part because conventional vapor-diffusion and microbatchprotocols do not readily allow for screening at anything typically otherthan atmospheric pressure. The rigidity of the PDMS matrix enablesexperiments to probe the effects of pressure on target crystallizationon-chip.

The surface on which the crystallization ‘drop’ sits can affectexperimental success and crystal quality. Examples of solid supportcontact surfaces used in vapor diffusion and microbatch protocolsinclude either polystyrene or silanized glass. Both types of supportscan show different propensities to promote or inhibit crystal growth,depending on the target. In addition, the crystallization ‘drop’ is incontact with either air or some type of poly-carbon oil, depending onwhether the experiment is a vapor-diffusion or microbatch setup,respectively. Air contact has the disadvantage in that free oxygenreacts readily with biological targets, which can lead to proteindenaturation and inhibit or degrade crystallization success. Oil allowstrace hydrocarbons to leach into the crystallization experiment, and cansimilarly inhibit or degrade crystallization success.

Microfluidics device designs in accordance with embodiments of thepresent invention may overcome these limitations by providing anonreactive, biocompatible environment that completely surrounds thecrystallization reaction. Moreover, the composition of thecrystallization chambers in the microfluidics chips can conceivably bevaried to provide new surfaces for contacting the crystallizationreaction; this would allow for routine screening of different surfacesand surface properties to promote crystallization.

Crystallization targets, particularly those of biological origin, mayoften be modified to enable crystallization. Such modifications includebut are not limited to truncations, limited proteolytic digests,site-directed mutants, inhibited or activated states, chemicalmodification or derivatization, etc. Target modifications can be timeconsuming and costly; modified targets require the same thoroughscreening as do unmodified targets. Microfluidics devices of the presentinvention work with such modified targets as readily as with theoriginal target, and provide the same benefits.

The effect of gravity as a parameter for crystallization is yet anotherunderstudied crystallization parameter, because of the difficulty ofvarying such a physical property. Nonetheless, crystallizationexperiments of biological samples in zero gravity environments haveresulted in the growth of crystals of superior quality than thoseobtained on Earth under the influence of gravity.

The absence of gravity presents problems for traditional vapor-diffusionand microbatch setups, because all fluids must be held in place bysurface tension. The need to often set up such experiments by hand alsoposes difficulties because of the expense of maintaining personnel inspace. Microfluidics devices in accordance with embodiments of thepresent invention, however, would enable further exploration ofmicrogravity as a crystallization condition. A compact, automatedmetering and crystal growth system would allow for: 1) launching ofsatellite factory containing target molecules in a cooled, but liquidstate, 2) distribution of targets and growth of crystals, 3) harvestingand cryofreezing of resultant crystals, and 4) return of cryo-storedcrystals to land-based stations for analysis.

8. In Situ Crystallization Screening

The ability to observe the growth of crystals with a microscope is astep in deciding upon success or failure of crystallization trials.Conventional crystallization protocols may use transparent materialssuch as polystyrene or silanized glass to allow for visualization. Thetransparency of the PDMS matrix of embodiments in accordance with thepresent invention is particularly suited to the two primary methods bywhich crystallization trials are traditionally scored: 1) directobservation in the visible light regime by optical microscopes and 2)birefringence of polarized light.

Birefringence may be difficult to judge in conventional experiments asmany plastics are themselves birefringent, interfering with sampleassessment. However, the microfluidics devices described herein can bemade without such optical interference properties, allowing for thedesign of an automated scanning system that routinely allows directvisualization with both polarizing and non-polarizing features.

In addition, robotic and, in particular, manually-set crystallizationexperiments can vary the placement of a crystallization drop on asurface by tens to hundreds of microns. This variability presents aproblem for automated scanning systems, as it is difficult to program inthe need for such flexible positioning without stable fiducials.However, the fixed placement of crystallization chambers in themicrofluidics chips of embodiments of the present invention overcomessuch problems, as every well can be positioned in a particular locationwith submicron accuracy. Moreover, such a system is readily scalable forthe design of differently sized and positioned crystallization chambers,as masks and other templates used to design microfluidics devices inaccordance with embodiments of the present invention can be simplydigitized and ported into scanning software for visualization.

Once crystals are obtained by visual inspection, it may be possible toscreen for diffraction directly through the chip itself. For example, acrystallization chamber within a chip could be outfitted withtransparent ‘windows’ comprising glass, quartz, or thinned portions ofthe elastomer material itself, on opposite walls of the chamber.Crystals could then be exposed directly to x-rays through the chip toassay for diffraction capabilities, eliminating the need to remove, andthereby possibly damage, the crystalline sample. Such an approach couldbe used to screen successes from initial crystallization trials todetermine the best starting candidate conditions for follow-up study.Similarly, crystals grown under a particular set of conditions could be‘re-equilibrated’ with new solutions (e.g., cryo-stabilizing agents,small-molecule drug leads or ligands, etc.), and the stability of thecrystals to such environment changes monitored directly by x-raydiffraction.

9. Utilizing Microfluidic Devices for Purification/Crystallization

Crystallization of target biological samples such as proteins isactually the culmination of a large number of prior complex anddifficult steps, including but not limited to protein expression,purification, derivatization, and labeling. Such steps prior tocrystallization comprise shuttling liquids from a chamber with one setof solution properties to another area with a different set ofproperties. Mircofluidics technology is suited to perform such tasks,allowing for the combination of all necessary steps within the confinesof a single chip.

Examples of microfluidic handling structures enabling performance ofpre-crystallization steps have been described under section I above. Forexample, a microfluidics chip could act as a regulated bioreactor,allowing nutrients to flow into growing cells contained in cell penstructure while removing wastes and inducing recombinantly-modifiedorganisms to produce target molecules (e.g., proteins) at a desiredstage of cell growth. Following induction, these cells could be shuntedfrom the cell pen to a different region of the chip for lysis byenzymatic or mechanical means. Solubilized target molecules could thenbe separated from cellular debris by molecular filtration unitsincorporated directly onto a chip.

The crude mixture of target molecules and contaminating cellularproteins and nucleic acids could then be funneled through porousmatrices of differing chemical properties (e.g., cation-exchange,anion-exchange, affinity, size-exclusion) to achieve separation. If atarget molecule were tagged with a fusion protein of a particular typeto promote solubility, it could be affinity purified, briefly treatedwith a similarly-tagged, site-specific protease to separate the fusionproduct, and then repassaged though the affinity matrix as a clean-upstep.

Once pure, the target could be mixed with different stabilizing agents,assayed for activity, and then transported to crystallization stagingareas. Localized heating (such as an electrode) and refrigeration (suchas a Peltier cooler) units stationed at various points on a chip or achip holder would allow for differential temperature regulation at allstages throughout the processing and crystallization. Thus, theproduction, purification, and crystallization of proteins may beaccomplished on an embodiment of a single microfluidics device inaccordance with the present invention.

IV. Micro-Free Interface Diffusion

A conventional approach to crystallization has been to effect a gradualchange in target solution conditions by introducing a crystallizingagent through slow diffusion. One method that is particularly effectiveat sampling a wide range of conditions is macroscopic free-interfacediffusion. This technique requires the creation of a well-definedfluidic interface between two or more solutions, typically the proteinstock, and the precipitating agent, and the subsequent equilibration ofthe two solutions via a diffusive process. As the solutions diffuse intoone another, a gradient is established along the diffusion path, and acontinuum of conditions is simultaneously sampled. Since there is avariation in the conditions, both in space, and in time, informationregarding the location and time of crystal formation may be used infurther optimization. FIGS. 29A-29D are simplified schematic diagramsplotting concentration versus distance for a solution A and a solution Bin contact along a free interface. FIGS. 29A-D show that over time, acontinuous and broad range of concentration profiles of the twosolutions is ultimately created.

Despite the efficiency of macroscopic free-interface diffusiontechniques, technical difficulties have rendered it unsuitable for highthroughput screening applications, and it is not widely used in thecrystallographic community for several reasons. First, the fluidicinterfaces are typically established by dispensing the solutions into anarrow container; such as a capillary tube or a deep well in a cultureplate. FIGS. 30A-B show simplified cross-sectional views of theattempted formation of a macroscopic free-interface in a capillary tube9300. The act of dispensing a second solution 9302 into a first solution9304 creates convective mixing and results in a poorly defined fluidicinterface 9306.

Moreover, the solutions may not be sucked into a capillary serially toeliminate this problem. FIGS. 31A-B show the mixing, between a firstsolution 9400 and a second solution 9402 in a capillary tube 9404 thatwould result due to the parabolic velocity distribution of pressuredriven Poiseuille flow, resulting in a poorly defined fluidic interface9406. Furthermore, the container for a macro free-interfacecrystallization regime must have dimensions making them accessible to apipette tip or dispensing tool, and necessitating the use of large(10-100 μl) volumes of protein and precipitant solutions.

In order to avoid unwanted convective mixing, care must be taken bothduring dispensing and during crystal incubation. For this reasoncumbersome protocols are often used to define a macro free-interface.For example, freezing one solution prior to the addition of the second.Moreover, two solutions of differing density will mix by gravity inducedconvection if they are not stored at the proper orientation,additionally complicating the storage of reactions. This is shown inFIGS. 32A-C, wherein over time first solution 9500 having a densitygreater than the density of second solution 9502 merely sinks to form astatic bottom layer 9504 that is not conducive to formation of adiffusion gradient along the length of a capillary tube.

In accordance with embodiments of the present invention, acrystallization technique analogous to traditional macro-free interfacediffusion, called gated micro free interface diffusion (Gated μ-FID),has been developed. Gated μ-FID retains the efficient sampling of phasespace achieved by macroscopic free interface diffusion techniques,

A microfluidic free interface (μFI) in accordance with embodiments ofthe present invention is a localized interface between at least onestatic fluid and another fluid wherein mixing between them is dominatedby diffusion rather than by convective flow. For the purposes of thisapplication, the term “fluid” refers to a material having a viscositybelow a particular maximum. Examples of such maximum viscosities includebut are not limited to 1000 CPoise, 900 CPoise, 800 CPoise, 700 CPoise,600 CPoise, 500 CPoise, 400 CPoise, 300 CPoise, 250 CPoise, and 100CPoise, and therefore exclude gels or polymers containing materialstrapped therein.

In a microfluidic free interface in accordance with an embodiment of thepresent invention, at least one dimension of the interface is restrictedin magnitude such that viscous forces dominate other forces. Forexample, in a microfluidic free interface in accordance with anembodiment of the present invention, the dominant forces acting upon thefluids are viscous rather than buoyant, and hence the microfluidic freeinterface may be characterized by an extremely low Grashof number (seediscussion below). The microfluidic free interface may also becharacterized by its localized nature relative to the total volumes ofthe fluids, such that the volumes of fluid exposed to the steeptransient concentration gradients present initially after formation ofthe interface between the pure fluids is limited.

The properties of a microfluidic free interface created in accordancewith embodiments of the present invention may be contrasted with anon-free microfluidic interface, as illustrated in FIGS. 33A and 33B.Specifically, FIG. 33A shows a simplified cross-sectional view of amicrofluidic free interface in accordance with an embodiment of thepresent invention. Microfluidic free interface 7500 of FIG. 33A isformed between first fluid A and second fluid B present within channel7502. The free microfluidic interface 7500 is substantially linear, withthe result that the steep concentration gradient arising between fluidsA and B is highly localized within the channel.

As described above, the dimensions of channel 7502 are extremely small,with the result that non-slip layers immediately adjacent to the wallsof the channel in fact occupy most of the volume of the channel. As aresult, viscosity forces are much greater than buoyant forces, andmixing between fluids A and B along interface 7500 occurs almostentirely as a result of diffusion, with little or no convective mixing.

Conditions associated with the microfluidic free interface ofembodiments of the present invention can be expressed in terms of theGrashof number (Gr) per Equation (3) below, an expression of therelative magnitude of buoyant and viscous forces:

$\begin{matrix}{{{{Gr} = {{B/V} = \frac{{\alpha\Delta}\;{cgL}^{3}}{v^{2}}}},{{where}\text{:}}}{{{Gr} = {{Grashof}\mspace{14mu}{number}}};}{{B = {{buoyancy}\mspace{14mu}{force}}};}{{V = {{viscous}\mspace{14mu}{force}}};}{{\alpha = {{solutal}\mspace{14mu}{expansivity}}};}{{c = {concentration}};}{{g = {{acceleration}\mspace{14mu}{of}\mspace{14mu}{gravity}}};}{{L = {{chamber}\mspace{14mu}{critical}\mspace{14mu}{dimension}}};}{and}{v = {{kinematic}\mspace{14mu}{{viscosity}.}}}} & (3)\end{matrix}$

According to Equation (3), a number of approaches may be taken to reducethe Grashof number and hence the presence of unwanted corrective flow.One such approach is to reduce g, and this is the tactic adopted bymicrogravity crystallization experiments conducted in space. Anotherapproach is to increase ν, and this is the tactic adopted byinvestigators working with gel acupuncture techniques, as describedgenerally by Garcia-Ruiz et al., “Agarose as Crystallization Media forProteins I: Transport Processes”, J. Crystal Growth 232, 165-172 (2001),hereby incorporated by reference for all purposes.

Embodiments in accordance with the present invention seek to reduce Land through the use of microfluid flow channels and vessels havingextremely small dimensions. The effect of this approach is amplified bythe cubed power of the variable (L) in Equation (3).

Microfluidic free interfaces in accordance with embodiments of thepresent invention would be expected to exhibit a Grashof number of 1 orless. The Grashof number expected with two fluids having the samedensity is zero, and thus Grashof, numbers very close to zero would beexpected to be attained.

The embodiment of a microfluidic free interface illustrated above inFIG. 33A may be contrasted with the conventional non-microfluidic freeinterface shown in FIG. 33B. Specifically, first and second fluids A andB are separated by an interface 7504 that is not uniform or limited bythe cross-sectional width of channel 7502. The steep concentrationgradient occurring at the interface is not localized, but is insteadpresent at various points along the length of the channel, exposingcorrespondingly large volumes of the fluids to the steep gradients. Inaddition, viscosity forces do not necessarily dominate over buoyancyforces, with the result that mixing of fluids A and B across interface7504 can occur both as the result of diffusion and of convective flow.The Grashof number exhibited by a conventional non-microfluidicinterface would be expected to exceed one.

1. Creation of Microfluidic Free Interface

A microfluidic free interface in accordance with embodiments of thepresent invention may be created in a variety of ways. One approach isto utilize the microfabricated elastomer structures previouslydescribed. Specifically, in certain embodiments the elastomeric materialfrom which microfluidic structures are formed is relatively permeable tocertain gases. This gas permeability property may be exploited utilizingthe technique of pressurized out-gas priming (POP) to form well-defined,reproducible fluidic interfaces.

FIG. 34A shows a plan view of a flow channel 9600 of a microfluidicdevice in accordance with an embodiment of the present invention. Flowchannel 9600 is separated into two halves by actuated valve 9602. Priorto the introduction of material, flow channel 9600 contains a gas 9604.

FIG. 34B shows the introduction of a first solution 9606 to first flowchannel portion 9600 a under pressure, and the introduction of a secondsolution 9608 to second flow channel portion 9600 b under pressure.Because of the gas permeability of the surrounding elastomer material9607, gas 9604 is displaced by the incoming solutions 9608 and 9610 andout-gasses through elastomer 9607.

As shown in FIG. 34C, the pressurized out-gas priming of flow channelportions 9600 a and 9600 b allows uniform filling of these dead-endedflow channel portions without air bubbles. Upon deactuation of valve9602 as shown in FIG. 34D, microfluidic free interface 9612 is defined,allowing for formation of a diffusion gradient between the fluids.

The formation of protein crystals utilizing gated μ-FID retains theefficient sampling of phase space achieved by macroscopic free interfacediffusion techniques, with a number of added advantages, including theparsimonious use of sample solutions, ease of set-up, creation of welldefined fluidic interfaces, control over equilibration dynamics, and theability to conduct high-throughput parallel experimentation.

Another possible advantage of the formation of protein crystalsutilizing gated μ-FID is the formation of high quality crystals, asillustrated in connection with FIGS. 36A and 36B. FIG. 36A shows asimplified schematic view of a protein crystal being formed utilizing aconventional macroscopic free interface diffusion technique.Specifically, nascent protein crystal 9200 is exposed to sample fromsolution 9202 that is experiencing a net conductive flow of sample as aresult of the action of buoyant forces. As a result of thedirectionality of this conductive flow, the growth of protein crystal9200 is also directional. However, as described in Nerad et al.,“Ground-Based Experiments on the Minimization of Convection During theGrowth of Crystals from Solution”, Journal of Crystal Growth 75, 591-608(1986), assymetrical growth of a protein crystal can give rise tounwanted strain in the lattice of the growing crystal, promotingdislocations and/or the incorporation of impurities within the lattice,and otherwise adversely affecting the crystal quality.

By contrast, FIG. 36B shows a simplified schematic view of a proteincrystal being formed utilizing diffusion across a microfluidic freeinterface in accordance with an embodiment of the present invention.Nascent protein crystal 9204 is exposed to sample solution 9206 that isdiffusing within the crystallizing agent. This diffusion isnondirectional, and the growth of protein crystal 9204 is alsocorrespondingly nondirectional. Accordingly, the growing crystal avoidsstrain on the lattice and the attendant incorporation of impurities anddislocations experienced by the growing crystal shown in FIG. 36A.Accordingly, the quality of the crystal in FIG. 36B is of high quality.

While the specific embodiment just described exploits the permeabilityof the bulk material to dead end fill two or more chambers or channelsseparated by a closed valve, and creates a microfluidic free interfacebetween the static fluids by the subsequent opening of this valve, othermechanisms for realizing a microfluidic free interface are possible.

For example, FIG. 46 shows one potential alternative method forestablishing a microfluidic free interface diffusion in accordance withthe present invention. Microfluidic channel 8100 carries fluid Aexperiencing a convective flow in the direction indicated by the arrow,such that static non-slip layers 8102 are created along the walls offlow channel 8100. Branch channel 8104 and dead-ended chamber 8106contain static fluid B. Because material surrounding the dead-endedchannel and chamber provide a back pressure, fluid B remains static andmicrofluidic free interface 8110 is created at mouth 8112 of branchchannel 8108 between flowing fluid A and static fluid B. As describedbelow, diffusion of fluid A or components thereof across themicrofluidic free interface can be exploited to obtain useful results.While the embodiment shown in FIG. 46 includes a dead-ended branchchannel and chamber, this is not required by the present invention, andthe branch channel could connect with another portion of the device, aslong as a sufficient counter pressure was maintained to prevent any netflow of fluid through the channel.

Another potential alternative method for establishing a microfluidicfree interface diffusion assay is the use of break-through valves andchambers. A break-through valve is not a true closing valve, but rathera structure that uses the surface tension of the working fluid to stopthe advance of the fluid. Since these valves depend on the surfacetension of the fluid they can only work while a free surface exists atthe valve; not when the fluid continuously fills both sides and theinterior of the valve structure.

A non-exclusive list of ways to achieve such a valve include but are notlimited to patches of hydrophobic material, hydrophobic treatment ofcertain areas, geometric constrictions (both in height and width) of achannel, geometric expansions (both in height and in width of achannel), changes in surface roughness on walls of a channel, andapplied electric potentials on the walls.

These “break-through” valves may be designed to withstand a fixed andwell defined pressure before they “break through” and allow fluid topass nearly unimpeded. The pressure in the channel can be controlled andhence the fluid can be caused to advance when desired. Different methodsof controlling this pressure include but are not limited to externallyapplied pressure at an input or output port, pressure derived fromcentrifugal force (i.e. by spinning the device), pressure derived fromlinear acceleration (i.e. applying an acceleration to the device with acomponent parallel to the channel), elecrokinetic pressure, internallygenerated pressure from bubble formation (by chemical reaction or byhydrolysis), pressure derived from mechanical pumping, or osmoticpressure.

“Break-through” valves may be used to create a microfluidic freeinterface as shown and described in connection with FIGS. 35A-E. FIG.35A shows a simplified plan view of a device for creating a microfluidicfree interface utilizing break through valves. First chamber 9100 is influid communication with second chamber 9102 through branches 9104 a and9104 b respectively, of T-shaped channel 9104.

First break through valve 9106 is located at outlet 9108 of firstchamber 9100. Second break through valve 9110 is located in branch 9104b upstream of inlet 9105 of second chamber 9102. Third break throughvalve 9112 is located at outlet 9114 of second chamber 9102.Breakthrough valves 9106, 9110, and 9112 may be formed from hydrophobicpatches, a constriction in the width of the flow channel, or some otherway as described generally above. In FIGS. 35A-E, an open break throughvalve is depicted as an unshaded circle, and a closed break throughvalve is depicted as a shaded circle.

In the initial stage shown in FIG. 35B, first chamber 9100 is chargedwith first fluid 9116 introduced through stem 9104 c and branch 9104 aof T-shaped channel 9104 and chamber inlet 9107 at a pressure below thebreak through pressure of any of the valves 9106, 9110, and 9112. In thesecond stage shown in FIG. 35C, second chamber 9102 is charged with abuffer or other intermediate fluid 9118 introduced through stem 9104 cof T-shaped channel 9104 and inlet 9105 at a pressure below the breakthrough pressure of valve 9106 but greater than the break throughpressures of valves 9110 and 9112.

In the third stage shown in FIG. 35D, intermediate fluid 9118 isreplaced in second chamber 9102 with second fluid 9120 introducedthrough stem 9104 c of T-shaped channel 9104 and inlet 9105 at apressure below the break through pressure of valve 9106 but greater thanthe break through pressures of valves 9110 and 9112. In the final stagedepicted in FIG. 35E, second fluid 9120 has replaced the intermediatefluid, leaving the first and second fluids 9116 and 9120 in separatechambers but fluidically connected through T-junction 9104, creating amicrofluidic free interface 9122.

The use of break through valves to create a microfluidic free interfacein accordance with embodiments of the present invention is not limitedto the specific example given above. For example, in alternativeembodiments the step of flushing with a buffer or intermediate solutionis not required, and the first solution could be removed by flushingdirectly with the second solution, with potential unwanted by-productsof mixing removed by the initial flow of the second solution through thechannels and chambers.

While the embodiments just described create the microfluidic freeinterface in a closed microfluidic device, this is not required byembodiments in accordance with the present invention. For example, analternative embodiment in accordance with the present invention mayutilize capillary forces to connect two reservoirs of fluid. In oneapproach, the open wells of a micro-titer plate could be connected by asegment of a glass capillary. The first solution would be dispensed intoone well such that it fills the well and is in contact with the glasscapillary. Capillary forces cause the first solution to enter and flowto the end of the capillary. Once at the end, the fluid motion ceases.Next, the second solution is added to the second well. This solution isin contact with the first solution at the capillary inlet and creates amicrofluidic interface between the two wells at the end of thecapillary.

The connecting path between the two wells need not be a glass capillary,and in alternative embodiments could instead comprise a strip ofhydrophilic material, for example a strip of glass or a line of silicadeposited by conventional CVD or PVD techniques. Alternatively, theconnecting paths could be established by paths of less hydrophobicmaterial between patterned regions of highly hydrophobic material.Moreover, there could be a plurality of such connections between thewells, or a plurality of interconnected chambers in variousconfigurations. Such interconnections could be established by the userprior to use of the device, allowing for rapid and efficient variationin fluidic conditions.

Where as in the previous example the two reservoirs are not enclosed bya microfluidic device but are connected instead through a microfluidicpath, an alternative embodiment could have reservoirs both enclosed andnot enclosed. For example, sample could be loaded into a microfluidicdevice and pushed to the end of an exit capillary or orifice (by any ofthe pressure methods described above). Once at the end of the exitcapillary, the capillary could be immersed in a reservoir of reagent. Inthis way, the microfluidic free interface is created between theexternal reservoir and the reservoir of reagent in the chip. This methodcould be used in parallel with many different output capillaries ororifices to screen a single sample against a plurality of differentreagents using microfluidic free interface diffusion.

In the example just described, the reagent is delivered from one or manyinlets to one or many different outlets “through” a microfluidic device.Alternatively, this reagent can be introduced through the same orificethat is to be used to create the microfluidic interface. Thesample-containing solution could be aspirated into a capillary (eitherby applying suction, or by capillary forces, or by applying pressure tothe solution) and then the capillary may be immersed in a reservoir ofcounter-reagent, creating a microfluidic interface between the end ofthe capillary and the reservoir. This could be done in a large array ofcapillaries for the parallel screening of many different reagents. Verysmall volumes of sample could be used since the capillaries can have afixed length beyond which the sample will not advance. Forcrystallization applications (see below), the capillaries could beremoved and mounted in an x-ray beam for diffraction studies, withoutrequiring handling of the crystals.

2. Reproducible Control Over Equilibration Parameters

One advantage of the use of microfluidic free interface diffusion inaccordance with embodiments of the present invention is the ability tocreate uniform and continuous concentration gradients that reproduciblysample a wide range of conditions. As the fluids on either side of theinterface diffuse into one another, a gradient is established along thediffusion path, and a continuum of conditions is simultaneously sampled.Since there is a variation in the conditions, both in space and time,information regarding the location and time of positive results (i.e.crystal formation) may be used in further optimization.

In many applications it is desirable to create a gradient of a conditionsuch as pH, concentration, or temperature. Such gradients may be usedfor screening applications, optimization of reaction conditions,kinetics studies, determination of binding affinities, dissociationconstants, enzyme-rate profiling, separation of macromolecules, and manyother applications. Due principally to the suppression of convectiveflow, diffusion across a microfluidic free interface in accordance withan embodiment of the present invention may be used to establish reliableand well-defined gradient.

The dimensional Einstein equation (4) may be employed to obtain a roughestimate of diffusion times across a microfluidic free interface.

$\begin{matrix}{{{t = \frac{x^{2}}{4D}};}{{where}\text{:}}{{t = {{diffusion}\mspace{14mu}{time}}};}{{x = {{longest}\mspace{14mu}{diffusion}\mspace{14mu}{length}}};}{and}{D = {{diffusion}\mspace{14mu}{coefficient}}}} & (4)\end{matrix}$Generally, as shown in Equation (5) below, the diffusion coefficientvaries inversely with the radius of gyration, and therefore as one overthe cube root of molecular weight.

$\begin{matrix}{{{D \propto \frac{1}{r} \propto \frac{1}{m^{1/3}}};}{{where}\text{:}}{{D = {{diffusion}\mspace{14mu}{coefficient}}};}{{r = {{radius}\mspace{14mu}{of}\mspace{14mu}{gyration}}};}{and}{m = {{molecular}\mspace{14mu}{weight}}}} & (5)\end{matrix}$

In reviewing equation (5), it is important to recognize that correlationbetween the radius of gyration (r) and the molecular weight (m) is onlyan approximation. Because of the dominance of viscous forces overinertial forces, the diffusion coefficient is in fact independent ofmolecular weight and is instead dependent upon the size and hence dragexperienced by the diffusing particle.

As compared with the rough 1.5 hr equilibration time for a dye, anapproximate equilibration time for a protein of 20 KDa over the samedistance is estimated to be approximately 45 hours. The equilibrationtime for a small salt of a molecular weight of 100 Da over the samedistance is about 45 minutes.

The relative concentrations resulting from diffusion across a fluidicinterface is determined not only by thermodynamic conditions exploredduring the equilibration, but also by the rate at which equilibrationtakes place. It is therefore potentially valuable to control thedynamics of equilibration.

In conventional macroscopic diffusion methods, only coarse control overthe dynamics of equilibration may be available through manipulation ofinitial conditions. For macroscopic free interface diffusion, oncediffusion begins, the experimenter has no control over the subsequentequilibration rate. For hanging drop experiments, the equilibration ratemay be changed by modifying the size of the initial drop, the total sizeof the reservoir, or the temperature of incubation. In microbatchexperiments, the rate at which the sample is concentrated may be variedby manipulating the size of the drop, and the identity and amount of thesurrounding oil. Since the equilibration rates depend in a complicatedmanner on these parameters, the dynamics of equilibration may only bechanged in a coarse manner. Moreover, once the experiment has begun, nofurther control over the equilibration dynamics is available.

By contrast, in a fluidic free interface experiment in accordance withan embodiment of the present invention, the parameters of diffusiveequilibration rate may also be controlled by manipulating dimensions ofchambers and connecting channels of a microfluidic structure. Forexample, in a microfluidic structure comprising reservoirs in fluidcommunication through a constricted channel, where no appreciablegradient exists in the reservoirs due to high concentrations orreplenishment of material, to good approximation the time required forequilibration varies linearly with the required diffusion length. Theequilibration rate also depends on the cross-sectional area of theconnecting channels. The required time for equilibration may thereforebe controlled by changing both the length, and the cross-sectional areaof the connecting channels.

For example, FIG. 40A shows a plan view of a simple embodiment of amicrofluidic structure in accordance with the present invention.Microfluidic structure 9701 comprises reservoirs 9700 and 9702containing first fluid A and second fluid B, respectively. Reservoirs9700 and 9702 are connected by channel 9704. Valve 9706 is positioned onthe connecting channel between reservoirs 9700 and 9702.

Connecting channel 9704 has a much smaller cross-sectional area thaneither of the reservoirs. For example, in particular embodiments ofmicrofluidic structures in accordance with the present invention, theratio of reservoir/channel cross-sectional area and thus the ratio ofmaximum ratio of cross-sectional area separating the two fluids, mayfall between 500 and 25,000. The minimum of this range describes a50×50×50 μm chamber connected to a 50×10 μm channel, and the maximum ofthis range describes a 500×500×500 μm chamber connected to a 10×1 μmchannel.

Initially, reservoirs 9700 and 9702 are filled with respective fluids,and valve 9706 is closed. Upon opening valve 9706, a microfluidic freeinterface in accordance with an embodiment of the present invention iscreated, and fluids A and B diffuse across this interface through thechannel into the respective reservoirs. Moreover, where the amount ofdiffusing material present in one reservoir is large and the capacity ofthe other reservoir to receive material without undergoing a significantconcentration change is also large, the concentrations of material inthe reservoirs will not change appreciably over time, and a steady stateof diffusion will be established.

Diffusion of fluids in the simple microfluidic structure shown in FIG.40 may be described by relatively simple equations. For example, the netflux of a chemical species from one chamber to the other may be simplydescribed by equation (6):

$\begin{matrix}{{{J = {D*A*\frac{\Delta\; C}{L}}};}{{where}\text{:}}{J = {{net}\mspace{14mu}{flux}\mspace{14mu}{of}\mspace{14mu}{chemical}\mspace{14mu}{species}}}{{D = {{diffusion}\mspace{14mu}{constant}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{chemical}\mspace{14mu}{species}}};}{{A = {{cross}\text{-}{sectional}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{connecting}\mspace{14mu}{channel}}};}{{{\Delta\; C} = {{concentration}\mspace{14mu}{difference}\mspace{14mu}{between}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{channels}}};}{and}{L = {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{connection}\mspace{14mu}{{channel}.}}}} & (6)\end{matrix}$

Following integration and extensive manipulation of the terms ofequation (6), the characteristic time τ for the equilibration of the twochambers, where one volume V₁ is originally at concentration C and theother volume V₂ is originally at concentration 0, can therefore be takento be as shown in Equation (7) below:

$\begin{matrix}{{{{\tau = {\frac{1}{{V_{1}/V_{2}} + 1}*\frac{1}{D}*\frac{L}{A/V_{1}}}};}{{where}\text{:}}{{\tau = {{equilibration}\mspace{14mu}{time}}};}V_{1} = \begin{matrix}{{volume}\mspace{14mu}{of}\mspace{14mu}{chamber}\;{initially}} \\{{{containing}\mspace{14mu}{the}\mspace{14mu}{chemical}\mspace{14mu}{species}};}\end{matrix}}{V_{2} = \begin{matrix}{{volume}\mspace{14mu}{of}\mspace{14mu}{chamber}\mspace{14mu}{into}\mspace{14mu}{which}} \\{{{the}\mspace{14mu}{chemical}\mspace{14mu}{species}{\;\mspace{11mu}}{is}\mspace{14mu}{diffusing}};}\end{matrix}}{{D = {{diffusion}\mspace{14mu}{constant}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{chemical}\mspace{14mu}{species}}};}{{A = {{cross}\text{-}{sectional}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{connecting}\mspace{14mu}{channel}}};}{and}{L = {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{connection}\mspace{14mu}{{channel}.}}}} & (7)\end{matrix}$

Therefore, for a given initial concentration of a chemical species in achamber of a defined volume, the characteristic equilibration timedepends in a linear manner from the diffusive length L and the ratio ofthe cross-sectional area to the volume (hereafter referred to simply asthe “area”), with the understanding that the term “area” refers to thearea normalized by the volume of the relevant chamber. Where twochambers are connected by a constricted channel, as in the structure ofFIG. 40A, the concentration drop from one channel to the other occursprimarily along the connecting channel and there is no appreciablegradient present in the chamber. This is shown in FIG. 40B, which is asimplified plot of concentration versus distance for the structure ofFIG. 40A.

The behavior of diffusion between the chambers of the microfluidicstructure of FIG. 40A can be modeled, for example, utilizing the PDEtoolbox of the MATLAB® software program sold by The MathWorks Inc. ofNatick, Mass. FIGS. 41 and 42 accordingly show the results of simulatingdiffusion of sodium chloride from a 300 um×300 um×100 um chamber toanother chamber of equal dimensions, through a 300 um long channel witha cross-sectional area of 1000 um. The initial concentrations of thechambers are 1 M and 0 M, respectively.

FIG. 41 plots the time required for the concentration in one of thereservoirs to reach 0.6 of the final equilibration concentration, versuschannel length. FIG. 41 shows the linear relationship between diffusiontime and channel length for this simple microfluidic system.

FIG. 42 plots the inverse of the time required for the concentration inone of the reservoirs to reach 0.6 of the final equilibrationconcentration (T_(0.6)), versus the area of the fluidic interfacecreated upon opening of the valve. FIG. 42 shows the linear relationshipbetween these parameters. The simple relationship between theequilibration time constant and the parameters of channel length and1/channel area allows for a reliable and intuitive method forcontrolling the rate of diffusive mixing across a microfluidic freeinterface in accordance with an embodiment of the present invention.

This relationship further allows for one reagent to be diffusively mixedwith a plurality of others at different rates that may be controlled bythe connecting channel geometry. For example, FIG. 38A shows three setsof pairs of compound chambers 9800, 9802, and 9804, each pair connectedby microchannels 9806 of a different length Δx. FIG. 38B plotsequilibration time versus equilibration distance. FIG. 38B shows thatthe required time for equilibration of the chambers of FIG. 38A variesas the length of the connecting channels.

FIG. 39 shows four compound chambers 9900, 9902, 9904, and 9906, eachhaving different arrangements of connecting microchannel(s) 9908.Microchannels 9908 have the same length, but differ in cross-sectionalarea and/or number of connecting channels. The rate of equilibration maythus be increased/decreased by decreasing/increasing the cross-sectionalarea, for example by decreasing/increasing the number of connectingchannels or the dimensions of those channels.

Another desirable aspect of microfluidic free interface diffusionstudies in accordance with embodiments of the present invention is theability to reproducibly explore a wide range of phase space. Forexample, it may be difficult to determine, a priori, which thermodynamicconditions will be favorable for a particular application (i.e.nucleation/growth of protein crystals), and therefore it is desirablethat a screening method sample as much of phase-space (as manyconditions) as possible. This can be accomplished by conducting aplurality of assays, and also through the phase space sampled during theevolution of each assay in time.

FIG. 43 shows the results of simulating the counter-diffusion oflysozyme and sodium chloride utilizing the microfluidic structure shownin FIG. 40, with different relative volumes of the two reservoirs, andwith initial concentrations normalized to 1. FIG. 43 presents a phasediagram depicting the phase space between fluids A and B, and the pathin phase space traversed in the reservoirs as the fluids diffuse acrossthe microfluidic free interface created by the opening of the valve inFIG. 40. FIG. 43 shows that the phase space sampled depends upon theinitial relative volumes of the fluids contained in the two reservoirs.By utilizing arrays of chamber with different sample volumes, and thenidentifying instances where diffusion across the channel yieldeddesirable results (i.e. crystal formation), promising starting pointsfor additional experimentation can be determined.

As described above, varying the length or cross-sectional area of achannel connecting two reservoirs changes the rate at which the speciesare mixed. However, so long as the channel volume remains small comparedas compared with the total reaction volume, there is little or no effecton the evolution of concentration in the chambers through phase space.The kinetics of the mixing are therefore decoupled from the phase-spaceevolution of the reaction, allowing the exercise independent controlover the kinetic and thermodynamic behavior of the diffusion.

For example, it is often desirable in crystallography to slow down theequilibration so as to allow for the growth of fewer and higher qualitycrystals. In conventional techniques this is often attempted by addingnew chemical constituents such as glycerol, or by using microbatchmethods. However, this addition of constituents is not wellcharacterized, is not always effective, and may inhibit the formation ofcrystals. Microbatch methods also may pose the disadvantage of lacking adriving force to promote continued crystal growth as protein in thesolution surrounding the crystal is depleted. Through the use ofdiffusion across a microfluidic free interface in accordance with anembodiment of the present invention, crystal formation may be slowed bya well-defined amount without altering the phase-space evolution, simplyby varying the width or cross-sectional area of the connecting channel.

The ability to control the rate at which equilibration proceeds hasfurther consequences in cases were one wishes to increase the totalvolume of a reaction while conserving both the thermodynamics and themicrofluidic free interface diffusion mixing. One such case arises againin the context of protein crystallography, in which an initial, smallvolume crystallization assay results in crystals of insufficient sizefor diffraction studies. In such a case, it is desirable to increase thereaction volume and thereby provide more protein available for crystalgrowth, while at the same time maintaining the same diffusive mixing andpath through phase space. By increasing the chamber volumesproportionally and decreasing the area of the channel, the area of theinterface relative to the total assay volume is reduced, and a largervolume would pass through the same phase space as in the original smallvolume conditions.

While the above description has focused upon diffusion of a singlespecies, gradients of two or more of species which do not interact witheach other may be created simultaneously and superimposed to create anarray of concentration conditions. FIG. 47 shows a plan view of oneexample of a microfluidic structure for creating such superimposedgradients. Flat, shallow chamber 8600 constricted in the verticaldirection is connected at its periphery to reservoirs 8602 and 8604having fixed concentrations of chemical species A and B, respectively.Sink 8606 in the form of a reservoir is maintained at a substantiallylower concentration of species A and B . After the initial transientequilibration, stable and well-defined gradients 8608 and 8610 ofspecies A and B respectively, are established in two dimensions.

As evident from inspection of FIG. 47, the precise shape and profile ofthe concentration gradient will vary according to a host of factors,including but not limited to the relative location and number of inletsto the chamber, which can also act as concentration sinks for thechemical species not contained therein (i.e. reservoir 8604 may act as asink for chemical species A). However, the spatial concentrationprofiles of each chemical species within the chamber may readily bemodeled using the MATLAB program previously described to describe atwo-dimensional, well-defined, and continuous spatial gradient.

The specific embodiment illustrated in FIG. 47 offers the disadvantageof continuous diffusion of materials. Hence, where diffusion of productsof reaction between the diffusing species is sought to be discerned,these products will themselves diffuse in the continuous gradient,thereby complicating analysis.

Accordingly, FIG. 48 shows a simplified plan view of an alternativeembodiment of a microfluidic structure for accomplishing diffusion intwo dimensions. Grid 8700 of intersecting orthogonal channels 8702establishes a spatial concentration gradient. Reservoirs 8704 and 8708of fixed concentrations of chemical species A and B are positioned onadjacent edges of grid 8700. Opposite to these reservoirs on the gridare two sinks 8703 of lower concentration of the chemical present in theopposing reservoirs.

Surrounding each channel junction 8710 are two pairs of valves 8712 and8714 which control diffusion through the grid in the vertical andhorizontal directions, respectively. Initially, only valve pairs 8714are opened to create a well-defined diffusion gradient of the firstchemical in the horizontal direction. Next, valve pairs 8714 are closedand valve pairs 8712 opened to create a well-defined diffusion gradientof the second chemical in the vertical direction. Isolated by adjacenthorizontal valves, the gradient of the first chemical species remainspresent in regions between the junctions.

Once the second (vertical) gradient is established, the two gradientscan be combined and by opening all the valve pairs for a short time toallow partial diffusive equilibration. After the period of diffusion haspassed, all the valve pairs are closed to contain the superimposedgradient. Alternatively, valve pairs 8712 and 8714 can be closed to haltdiffusion in the vertical direction, with every second horizontal valveopened to create separate isolated chambers.

To summarize, conventional macro free-interface techniques employcapillary tubes or other containers having dimensions on the order ofmm. By contrast, the fluidic interface in accordance with embodiments ofthe present invention is created in a microchannel having dimensions onthe order of μm. At such small dimensions, unwanted convection issuppressed due to viscosity effects, and mixing is dominated bydiffusion. A well-defined fluidic interface may thus be establishedwithout significant undesirable convective mixing.

V. Exploration of Phase Space

Closely related to the problem of protein crystallography is determiningthe solubility of a protein as a function of several chemical variables.Since it may be difficult to determine, a priori, which thermodynamicconditions will induce crystallization, a screening method should sampleas much of phase-space (as many conditions) as possible. This can beaccomplished by conducting a plurality of assays, and also through thephase space sampled during the evolution of each assay in time.

The mixing and metering functionality of microfluidic devices andmethods in accordance with the present invention is suited to this task,whereby a protein sample may be mixed with a plurality of relatedsolutions whose chemistry is systematically varied. It is possible touse the universal phase properties of the precipitant proteininteraction to systematically design experiments that increase thechances of achieving crystal growth. In this way a solubility“phase-space” may be generated. The knowledge of this phase space may beused predict successful crystallization conditions or to refineidentified conditions.

FIG. 55 is a simplified schematic diagram showing a phase space of amixture comprising a macromolecule and a precipitating agent. This phasespace is divided by solubility curve 5500 into soluble (S) andsupersaturation (SS) regions determined by macromolecule solubility.

A Gibbs free energy diagram graphically represents the relative energiesof soluble and precipitation phases, separated by a barrier energy(E_(b)) required to move from the energetically disfavored to theenergetically favored state. FIG. 56A is a free energy diagram ofsoluble region (S) of FIG. 55. FIG. 56B is a free energy diagram alongsolubility curve 5500. FIG. 56C is a free energy diagram ofsupersaturated region (SS) of FIG. 55. Comparison of FIGS. 56A-C showenergies of the soluble and precipitate phases to be evenly balancedalong the solubility curve, with the soluble form energetically favoredin the soluble region and the solid phase energetically favored in thesupersaturated region.

FIG. 55 also shows that supersaturation region (SS) may further bedivided into a precipitation region (P) where amorphous precipitatelacking long range order (i.e. noncrystalline) forms rapidly, a labileregion (L) near the precipitation curve 5502, and a metastable region(M) near the solubility curve 5500. FIGS. 56D, 56E and 56F show freeenergy diagrams for the precipitation, labile, and metastable regions,respectively.

In the precipitation region (P), amorphous aggregate is favored overcrystalline solid, and the activation energy (E_(b)) is low, so that thetransition between soluble and solid states occurs rapidly. In thelabile region (L) the crystalline form is favored, but E_(b) is low,resulting in rapid nucleation and the corresponding formation of manysmall crystals. By contrast, in the metastable region (M) the relativelyhigh activation energy suppresses nucleation but supports growth ofexisting crystals.

Since the three dimensional nucleation required for critical nucleusaggregation generally has a larger activation energy than that ofsubsequent one or two dimensional nucleation needed for crystal facetgrowth, an optimal crystal growth scheme should provide independentcontrol over the these two phases of crystal growth. The BIM meteringscheme provides exactly this property by implementing “free interfacediffusion” between the precipitant and the protein solutions.

FIG. 57 is a simplified schematic diagram contrasting the evolutionthrough a two-dimensional phase space having macromolecule concentrationand precipitating agent concentration as variables, of conventionalhanging drop and microbatch experiments, and μFID experiments inaccordance with embodiments of the present invention. The phase-spacetrajectory taken by the chip during equilibration depends on thediffusion constants of the species involved. A short time after the chipinterface valves are opened, the protein concentration on the proteinside changes very little while that of the counter solvent, whichtypically has a much larger diffusion constant, increases to one ofthree final values determined by the lithographically defined mixingratios. Subsequently, over a time of approximately 8-24 hours theprotein concentration equilibrates, increasing on the solvent side anddecreasing on the protein side. The final protein concentration is onceagain determined by the mixing ratios. The chip therefore takes a curvedpath through phase space, which in principle allows the protein solutionto have efficient crystal nucleation in the labile region followed byhigh quality growth in the metastable region.

Accordingly, FIG. 57 shows evolution of a μFID reaction site havingthree different mixing ratios. Curves represent the average state ofboth the sample side and precipitating agent side of each compound well.The final states (I, II, III) are determined by the mixing ratio, andone can see that these curves have a greater chance of passing fromregions with a high probability of nucleation to a region that supportshigh quality crystal growth. The break points of the curves aredetermined by the mixing ratios.

By contrast, the conventional micro batch and hanging drop approachesstart at point IV where the target molecule is combined 1:1 with theprecipitating agent. Microbatch experiments are incubated underimmiscible oil, preventing subsequent concentration of reagents andtherefore sampling only a single point in phase space.

In hanging drop experiments, the mixture is allowed to equilibratethrough vapor diffusion with a large reservoir of precipitating agent,slowly concentrating the reagents and driving the sample into the supersaturation region. This is undesirable because the resulting phase spacetrajectory moves into the precipitating region.

While the use of free interface diffusion techniques offers a promisingway of sampling phase space, the sheer number and concentrations ofpotential crystallizing agents for any given macromolecule makes moresystematic and rapid phase space mapping techniques desirable.

For example, before designing an experiment or a set of experiments toinvestigate crystal growth, it may first prove more efficient toidentify the location of a curve for a particular combination ofmacromolecules and crystallizing agents. Once such a solubility curve ismapped in phase space, the investigator can then proceed to efficientlydesign a set of screening experiments in which the trajectory would beexpected traverse regions promising phase space regions adjacent to thissolubility curve.

The level of supersaturation of a macromolecule is generally defined byEquation (8) below:

$\begin{matrix}{{{{SS} = {\frac{{PC}\text{-}{MC}}{MC}*100}},{{where}\text{:}}}{{{SS} = {{level}\mspace{14mu}{of}\mspace{14mu}{supersaturation}\mspace{14mu}{of}\mspace{14mu} a\mspace{14mu}{macromolecule}}};}{{{PC} = {{macromolecule}\mspace{14mu}{concentration}}};}{and}{{MC} = \begin{matrix}{{Maximum}\mspace{14mu}{soluble}\mspace{14mu}{macromolecule}} \\{{concentration}\mspace{14mu}{in}\mspace{14mu}{{equilibrium}.}}\end{matrix}}} & (8)\end{matrix}$

For purposes of the instant invention, an alternative measure of thesupersaturation of a macromolecule, hereto referred to as the immediatesuper saturation (ISS), is obtained if MC is replaced by the immediatemaximum macromolecule concentration of protein (IMC). For the purposesof this invention, the IMC is defined as the maximum concentration ofmacromolecule that fails to produce a solid phase (either crystalline oramorphous), within 1 minute or less. The immediate supersaturation (ISS)is defined by Equation (9) below:

$\begin{matrix}{{{{ISS} = \frac{{PC}\text{-}{IMC}}{IMC}},{{where}\text{:}}}{{{ISS} = {{immediate}\mspace{14mu}{supersaturation}}};}{{{PC} = {{macromolecule}\mspace{14mu}{concentration}}};}{and}{{IMC} = {{Immediate}\mspace{14mu}{maximum}\mspace{14mu}{macromolecule}\mspace{14mu}{{concentration}.}}}} & (9)\end{matrix}$

Crystallization of a macromolecule generally requires highsupersaturation values typically in the range of 50% to 500%.Furthermore, it is generally undesirable to begin at positive ISS valuesin a crystallization experiment, as since this will result in immediateformation of a solid phase.

In a given phase space, the area bounded above by the IMC curve, andbounded below by the MC curve, defines a region in which protein crystalgrowth may be supported. Therefore if the IMC curve is known, forexample by observation of rapid solid formation during high throughputscreening utilizing combinatoric mixing, a crystallization experimentshould be set to evolve near the IMC and with negative ISS values. Forthe purpose of this patent application, conditions having ISS valuesbetween about ±50% are considered near the IMC curve.

The combinatoric mixing device previously discussed in connection withFIGS. 17A-B, and variants thereof, offer a rapid and effective way tomap the solubility curve of a crystallizing agent and a macromolecule.Specifically, the crystallizing agent sample could rapidly be preparedby injecting buffer and reagent into the rotary mixer flow channel toachieve a specific and precise concentration. Next, the sample injectionport line, and pump could be used to introduce the macromolecule sampleinto the rotary mixer, followed by detection of formation of precipitatein the rotary mixer.

Detection of precipitate/crystal aggregation may be done in severalways. One method of detection aggregation is to image the mixing ringonto a camera. Simple image processing may then be done to distinguish aclear channel from one having particulates. Additionally, since somecrystals show a degree of birefringence, a polarizing lens may be usedto distinguish crystalline from amorphous solid. It should also bepossible to use methods such as light scattering to detect the proteinaggregates on smaller length scales.

FIG. 24 shows a solubility “phase space” for a protein sample (Lysosyme84 mg/ml) being titrated against a salt solution (3.6 M NaCl, and 100 mMsodium acetate (NaAc) @ pH 4.6). shows the results of 100 assays,consuming a total volume of 120 mL. FIG. 24 represents only one ofthousands of such graphs comprising a solubility phase-space.

The graph shown in FIG. 24 illustrates only the distinction betweensoluble and precipitating conditions. However, it may be possible toobtain more information by shifting the direction of change in relativeconcentration of macromolecule and crystallizing agent, to pass throughthe solubility curve in both directions. For example, if enough salt isadded to a protein sample, it will form a solid that may be eitheramorphous (precipitate) or crystalline (micro-crystals). If thissolution is slowly diluted, the solid will eventually dissolve back intosolution. The concentration at which it dissolves occurs will, however,not typically be at the same concentration at which solidificationoriginally occurred, and will generally be dependant upon whether thesolid is crystalline or amorphous.

Specifically, while precipitation occurs nearly instantaneously,crystals take longer to form, suggesting a higher activation energy forthe crystallization process. The higher activation energy for crystalnucleation/formation implies that observation of nucleation on alaboratory timescale requires substantial supersaturation. In contrast,the crystal form, once it appears, is favored over the soluble form forall supersaturation values greater than zero. Thus the magnitude of thehysteresis under conditions where the soluble phase is converted tosolid, contrasted with conditions under which the solid phase isreconverted back to soluble phase, could reveal the presence of crystalsversus precipitate, and thus conditions favorable to crystallization.

FIG. 25 shows the precipitation of a sample of Lysozyme (84 mg/ml)precipitated by mixing with a crystallizing agent of (3.6M NaCl, and0.11 M sodium citrate @ pH 4.6). Lack of coincidence between square (▪)and circle (●) symbols indicates a hysteresis and a point for furtherpromising exploration of phase space to identify crystal growth.

Detection of a hysteresis in precipitation formation as just describedmay also serve to prove extremely valuable for identifying whether ornot a particular macromolecule/crystallizing agent combination holdspromise of forming crystals at all. For example, inspection of FIG. 55indicates that the labile and metastable supersaturation regionsfavorable to crystal formation do not occur uniformly along thesolubility curve. Instead, such labile/metastable regions may belocalized and surrounded by adjacent precipitation regions whereinformation of a solid material is amorphous and formation of orderedcrystalline material is not possible. Detection of a hysteresis inaccordance with an embodiment of the present invention may reveal theformation of some type of crystal, and thus provide a preliminaryscreening mechanism to minimize the time and effort required to mappotentially favorable regions of the phase space

It may further be possible to utilize light scattering techniques todirectly measure the size of aggregated solids in a crystallizationsample. Specifically, the virtual transparency of the PDMS of the chipto forms of incident electromagnetic radiation would enable opticalinterrogation of the flow channel of the rotary or other type of mixingdevice. Detection of radiation scattered from the sample utilizingtechniques such as quasi-elastic light scattering (QELS) or dynamiclight scattering (DLS) would enable the determination of the size ofsolid present in the sample, thereby allowing for determination ofsample conditions at the onset of solid formation, when crystalnucleation may be favored.

Moreover, in “Predicting Protein Crystallization From a Dilute SolutionProperty”, Acta Crystallogr. D. 50:361-365 (1994), George and Wilsondemonstrated a relationship between protein crystallization behavior andthe protein osmotic second virial coefficient (B₂₂), a basic parameterrepresenting the integral of the intermolecular potential over distance.The George and Wilson article is incorporated in its entirety herein forall purposes.

The second virial coefficient of a macromolecule solution may bedetected from the scattering behavior of a macromolecule solution.Moreover, the value of the second virial coefficient of proteinsolutions giving rise to crystallization has been found to lie in auniversally narrow range. Thus on-chip evaluation of the second virialcoefficient by light scattering techniques coupled with the ability toperform rapid combinatoric mixing with small volume samples would enablethe rapid screening of different mixtures for potentialcrystallizability.

Systematic Investigation of Protein Phase Behavior with a MicrofluidicFormulator

The application of x-ray crystallography to the determination of proteinstructure with atomic resolution was a triumph of structural biology inthe 20^(th) century. Since the first solution of the structure ofmyoglobin in 1958 by Kendrew et al., Nature 181, 662-666 (1958),incorporated by reference herein for all purposes, over 23,000 differentstructures have been deposited in the protein data bank, and their rolein relating structure to function in biology has been profound.

Structure determination efforts continue to move past the most tractablecrystallization targets (typically small soluble proteins), and focusinstead on more challenging macromolecules such as large proteincomplexes and membrane proteins. See Loll, Journal of Structural Biology142, 144-153 (2003), incorporated by reference herein for all purposes.Therefore, the need to better understand and explore the crystallizationprocess has become urgent. That is because once high quality crystalsare in hand, advances in x-ray sources, computer codes, and relatedtechnology have made it relatively straightforward to obtain thestructure. However, these innovations have not been matched bytechniques for rapidly expressing, purifying, and crystallizingproteins. As described by Chayen et al., Acta Crystallographica SectionD Biological Crystallography 58, 921-927 (2002), incorporated byreference herein for all purposes, determining the appropriatecrystallization conditions has become one of the most significantremaining bottlenecks to structure determination.

Understanding the phase behavior of proteins is a part of thecrystallization process. The growth of crystals from a protein solutionrequires the existence of a nontrivial phase diagram which allows theprotein state to be manipulated between at least two thermodynamicphases: soluble and precipitated. The processes of crystal nucleationand growth arise on the boundary between these two phases, and aregoverned by subtle effects in physical chemistry.

There are a variety of schemes that manipulate the kinetics of thecrystallization process, and all take advantage of generic features ofthese phase diagrams. See Luft et al., Macromolecular Crystallography,Pt A, Vol. 276, pp. 110-131 (1997), incorporated by reference herein forall purposes. However, in practice the phase behavior of very fewproteins has been studied in detail. See, e.g., Rosenbaum et al.,Journal of Crystal Growth 169, 752-758 (1996); Ataka, Phase Transitions45, 205-219 (1993); Carbonnaux et al., Protein Science 4, 2123-2128(1995); Mikol et al., Journal of Crystal Growth 97, 324-332 (1989);Howard et al., Journal of Crystal Growth 90, 94-104 (1988); Kam et al.,Journal of Molecular Biology 123, 539-555 (1978); Muschol et al.,Journal of Chemical Physics 107, 1953-1962 (1997); Forsythe et al.,Journal of Chemical and Engineering Data 44, 637-640 (1999), each ofwhich is incorporated by reference herein for all purposes. In addition,solubility information for a specific protein is rarely available forcrystallization and optimization experiments. See, e.g., Saridakis etal., Acta Crystallographica Section D-Biological Crystallography 50,293-297 (1994); and Saridakis et al., N. E. Biophysical Journal 84,1218-1222 (2003), incorporated by reference herein for all purposes.

Furthermore, it is often an arduous process to find the rightcombination of chemicals that yields appropriate phase behavior for agiven protein. Every protein is different, and even a modest subset ofstock precipitating solutions comprise a vast chemical phase space thatmust be explored. The large amounts of sample required make systematicexploration by conventional techniques infeasible, and screening istypically directed towards an incomplete factorial or sparse-matrixapproach, which is a brute-force process requiring large numbers ofexperiments. See Carter et al., J. Cryst. Growth 90, 60-73 (1988); andJancarik et al., J. Appl. Crystallogr. 24, 409-411 (1991), incorporatedby reference herein for all purposes.

There have been numerous attempts to rationalize this procedure. Oneapproach is to use computational approaches to predict phase behavior.See Carter et al., and Jancarik et al. Another approach is to try tocorrelate measurements of osmotic 2^(nd) virial coefficients withcrystallization conditions. See George et al., Acta CrystallographicaSection D-Biological Crystallography 50, 361-365 (1994); Guo et al.,Journal of Crystal Growth 196, 424-433 (1999), incorporated by referenceherein for all purposes. Practical limitations have thus far preventedthese techniques from being generally applicable to the determination ofcrystallization conditions.

Here we describe a microfluidic formulation device that allows for thecombinatorial mixing of 16 buffers and 16 precipitation agents with apurified protein sample. The ability of the formulation chip to access avast number of chemical conditions, and to accurately dispense and mixfluids on the picoliter scale makes detailed characterization ofmacromolecule phase behavior both possible and practical. We used thisdevice to screen 5,000 different solubility conditions of the modelprotein Endo-1,4-β-xylanase from Trichoderma reesei. Xylanase is a 21KDa member of the gluconase enzyme family.

For those conditions that exhibited non-trivial phase behavior (ieprecipitation), a full phase diagram was generated. From this thoroughcharacterization of the phase behavior, we designed a rationalcrystallization screen for xylanase. Comparison of this screen to 4commercially available sparse matrix screens showed nearly two orders ofmagnitude increase in crystallization success, and allowed new insightinto the physics of crystallization.

Samples were prepared and crystallization protocols followed, as setforth below. Endo-1,4-β-xylanase (xylanase) from Trichoderma reesei(Hampton Research) was prepared in deionized water from stock (36 mg/mLprotein, 43% wt/vol glycerol, 0.18 M sodium/potassium phosphate pH 7.0)by repeated buffer exchange at 4° C. using a centrifugal filter with amolecular weight cut-off of 10,000 Da (Micon Bioseparations). Proteinconcentration was measured by absorption at 280-nm and adjusted to 120mg/mL. 10 μL aliquots were flash frozen in liquid nitrogen and stored at−80° C. To avoid sample-sample variations, a single sample preparationwas used for all solubility screening, phase space mapping andcorresponding crystallization experiments.

Batch crystallization trials were actively mixed by repeated aspirationand incubated under paraffin oil. Crystallization trials were inspecteddaily for a period of two weeks. Observed crystals were confirmed to beprotein crystals by staining (IZIT dye; Hampton Research) and wererecorded as crystallization hits.

All photo-masks for the master model were designed using AutoCAD(Autodesk) and printed at a resolution of 20,000 dpi on a transparencyfilm (CAD/Art Services). The flow-layer master was fabricated from acombination of positive and negative photoresists using a three-steplithography process. 9 μm high channel sections defining the top andbottom of the mixing ring structure were fabricated from SU8-2010resist. These features provide a channel section with well-definedrectangular cross-section that does not reflow during subsequentprocessing, thereby facilitating absorption and precipitationmeasurements.

SU8 2010 (MicroChem) was spun onto a silicon wafer (3,000 rpm for 45seconds), pre-exposure baked (1 minute 65° C./3 minutes 95° C.), exposedthrough a negative transparency mask (40 seconds 7 mW/cm²),post-exposure baked (1 minute 65° C./3 minutes 95° C.), and developed inSU8 nano developer (MicroChem).

Channel sections compatible with integrated valves were fabricated usingSJR 5740 positive photoresist (Shipley). To promote photoresist adhesionthe wafer was first treated with hexamethyldisilazane (MicroprimeHP-Primer; ShinEtsu MicroSi) (1 minute at 1 atmosphere). Photoresist wasspun onto the patterned wafer (2,000 rpm for 60 seconds), soft baked (1minute 45 seconds 95° C.), aligned to the existing features, exposed (45seconds/seconds 7 mW/cm²), and developed (20% Microposit 2401 developer;Shipley). The mold was then annealed (20 minutes/120° C.), resulting ina smooth rounded cross-section necessary for valve closure, and hardbaked (2 hours/170° C.).

Low impedance input and output channels were fabricated to allow for therapid flushing of viscous reagents. A 60 μm layer of SU8 2075(MicroChem) was spun onto a silicon wafer (3,000 rpm for 60 seconds),pre-exposure baked (7 minute 65° C./20 minutes 95° C.), aligned to theprimary flow structure, and exposed through a negative transparency mask(40 seconds 7 mW/cm²), post-exposure baked (1 minute 65° C./15 minutes95° C.), and developed in SU8 nano developer (MicroChem). 25 μm highcontrol features were fabricated on a separate wafer using a singlelithographic step. SU8 2025 (MicroChem) was spun onto a silicon wafer(3,000 rpm for 45 seconds), pre-exposure baked (1 minute 65° C./3minutes 95° C.), aligned to the primary flow structure, and exposedthrough a negative transparency mask (40 seconds 7 mW/cm2),post-exposure baked (1 minute 65° C./3 minutes 95° C.), and developed inSU8 nano developer (MicroChem).

Microfluidic devices were fabricated as follows. The microfluidicformulator was fabricated from silicone elastomer (General Electric RTV615) using the technique of multilayer soft lithography of Unger et al.,Science 288, 113-116 (2000), incorporated by reference herein for allpurposes. To facilitate the release of the elastomer from the mold allmolds were treated with chlorotrimethylsilane (Aldrich). Consecutivereplica molding from microfabricated masters and chemical bonding stepswere used to create a three-layer elastomer device consisting of a 7 mmthick layer with patterned flow structure (top), a 35 μm control layer(middle), and a featureless sealing layer (bottom).

Liquid silicone elastomer (20 part A:1 part B) was spun onto the controlmaster (2400 rpm for 60 seconds) and baked in a convection oven at 80°C. for 60 minutes. Liquid silicone elastomer (5 part A:1 part B) waspoured on the flow master to a thickness of 7 mm, degassed, and baked at80° C. for 75 minutes.

The partially cured flow layer was peeled from the master and aligned tothe control mold. The two-layer structure was then baked for 75 minutes,chemically cross-linking the two layers into a single structure. Thebonded elastomer was then peeled from the control mold and access portswere punched at the flow and control inlets using a 0.055 inch punch(Technical Innovations).

The structure was then placed on a featureless elastomer membrane (20part A:1 part B) created by spinning elastomer on a plain silicon waferat 2500 rpm for 1 minute and baking for 1 hour at 80° C. The assembledstructure was then baked overnight, causing the three layers to bondinto a monolithic multilayer device. Finally the device was peeled fromthe silicon wafer, cut to size, and sealed to a glass substrate formechanical rigidity.

Experimental setup and data collection were performed as follows.Automation of metering, mixing and data acquisition allows for thousandsof solubility experiments to be executed without the need for userintervention. In each solubility experiment a unique mixture of the 32reagents and the protein sample is produced.

All device control and data acquisition was implemented using a customsoftware driver developed in LabView (National Instruments). Mixingrecipes were generated using a spread-sheet program and translated intovalve actuation sequences by the software driver. Off-chip solenoidvalves (Lee Products Ltd.), controlled using a digital input output card(DIO-32HS; National Instruments), were used to generate square-wavepressure signals at the device control ports. A frame-grabber card(Imagenation PXC200A; CyberOptics) was used to automate imageacquisition from a charge coupled device camera.

The on-chip peristaltic pumps were pneumatically actuated at 100 Hz,resulting in a maximum flow velocity of approximately 2 cm/s. At theseflow rates complete mixing of aqueous reagents was achieved in less then3 seconds, and solutions with viscosities of approximately 100 cP weremixed in 6 seconds.

Absorption and precipitation measurements were taken as follows.Absorption measurements were taken to determine the concentration ofbromophenol blue sodium salt (absorption peak at 590-nm) in the mixingring. A 9 μm high segment of the mixing ring (approximately 300 μm by 80μm) having rectangular cross-section was illuminated with a 590-nm diode(AND180HYP; Newark Electronics) and imaged through a stereoscope (SMZ1500; Nikon) onto a charge coupled device camera. Pixel intensities wereaveraged and compared to an identical adjacent reference channelcontaining the undiluted dye (2 mM bromophenol blue sodium salt, 100 mMTRIS-HCl pH 8.0). In some experiments glycerol was added to the injecteddye to vary the viscosity. Dye concentrations were determined using theBeer-Lambert relation and used to calculate the injected volume.

Precipitation of the protein was automatically detected by imaging aportion of the mixing ring, calculating the standard deviation of thepixel intensities and comparing this value to the background (no proteinadded). To ensure even illumination, images were taken at 112 timesmagnification at a 9 μm high section of the mixing ring havingrectangular cross-section.

The positive-displacement cross-injection metering scheme allows forsequential injection of precise sample aliquots from a singlemicrofluidic channel into an array of reaction chambers through apositive displacement cross-injection (PCI) junction. FIGS. 62A-D showsimplified schematic views of positive displacement cross-injection(PCI) for robust and programmable high precision dispensing on chip.

FIG. 62A shows a schematic view of a four port PCI junction. As shown inFIG. 62A, the PCI junction 6200 is formed by the combination of athree-valve peristaltic pump 6202 and a novel four-port cross-injectionjunction with integrated valves on each port. At each junction, two setsof valves 6204 and 6206 are actuated to direct the flow eitherhorizontally or vertically. The split channel architecture creates alarger volume injector region, thereby allowing for an increased numberof injections before recharging.

FIG. 62B shows charging the injector region of the PCI junction. Toexecute the metering task, the flow is switched vertically through thejunction, charging the cross-injector with the sample fluid. Junctionvalves are actuated to direct the flow vertically through the junction,filling the injector region.

FIG. 62C shows precise positive displacement metering by actuation ofperistaltic pump valves in pumping sequence. The flow is then directedhorizontally through the junction and the three valves forming theperistaltic pump are actuated in a five state sequence to advance thefluid in the horizontal direction.

FIG. 62D shows the PCI junction sequentially charged with differentsolutions to create complex multi-component mixtures. Each cycle of theperistaltic pump injects a well-defined volume of sample (approximately80 pL), determined by the dead volume under the middle valve of theperistaltic pump. The deflection of the valve membranes when notactuated is determined by the pressure difference across the membrane.The volume injected during each cycle therefore may be tunedcontinuously, allowing for variable positive displacement metering. Byrepeating the injection sequence, the volume of injected solution may beincreased in 80 pL increments, allowing for the dynamic quantizedcontrol of the final downstream sample concentration.

The dearth of available information regarding protein solubility may belargely attributed to practical limitations of conventional fluidhandling technology. Although small scale characterization of proteinsolubility by a pre-crystallization solubility assay has been reportedby Stura et al., Journal of Crystal Growth 122, 273-285 (1992) and bySantesson et al., Analytical Chemistry 75, 1733-1740 (2003), both ofwhich are incorporated by reference herein for all purposes, thistechnique has not been widely adopted since the large required samplevolumes make it unsuitable for targets that cannot be expressed andpurified in large quantities. Microfabricated dispensers have been usedto reduce sample consumption in cases where the sequential addition ofreagents to a levitated drop of microliter volume is sufficient toexplore a restricted chemical space (Santesson et al). While micofluidicdevices have been previously used to screen crystallization conditionsusing free interface diffusion by Hansen et al., Proc. Nat'l. Academy ofSciences 99, 16531-16536 (2002) and microbatch formats by Zheng et al.,Journal of the American Chemical Society 125, 11170-11171 (2003), bothof which are incorporated by reference herein for all purposes, theyhave not been applied to systematically measure phase behavior. Previouslimitations in fluid handling functionality have limited the use ofmicrofluidic devices in applications such as protein phase space mappingwhich may involve the complex on-chip mixing of reagents.

Thorough characterization of protein solubility behavior involvesaccessing chemical space through the combinatorial mixing of a limitednumber of stock reagents. The conventional reagents used incrystallization exhibit a large variation in physical properties such asviscosity, surface tension, ionic strength, and pH. This variationpresents a formidable challenge for fluid handling systems that mustallow for arbitrary fluid combinations and proportioning.

We developed a positive displacement cross-injection metering methodthat overcomes this obstacle, allowing for variable dispensing to bedynamically programmed by the user in 80 picoliter increments with lessthan 5% variation over a broad range of fluid properties. By combiningthis method with microfluidic mixing, Chu et al., BiomedicalMicrodevices 3, 323-330 (2001), incorporated by reference herein for allpurposes, and multiplexing elements, Thorsen et al., Science 298,580-584 (2002), incorporated by reference herein for all purposes, largescale combinatorial screening has been achieved on chip for the firsttime. The flexibility, precision and small volume requirements of thisdevice make feasible the systematic mapping of crystallization phasespace.

FIGS. 63A-D show combinatorial mixing using a microfluidic formulator.FIG. 63A shows integration of the multiplexer, peristaltic pumps, rotarymixer, and PCI junction components for on-chip combinatorialformulation.

FIG. 63B shows injection of approximately 250 pL (4 injection cycles) ofdye into rotary mixer. FIG. 63C shows the color gradient formed byconsecutive injections into the mixing ring (8 injections blue, 8injections green, 8 injections yellow, 8 injections red). FIG. 63D showspumping around the ring for 3 seconds results in complete mixing of dye.Blue dye is added to mixture through sample injection inlet (bottomright).

The active region of microfluidic formulation chip that implements thisscheme and allows for the arbitrary combinatorial mixing of 16 stockreagents into one of 16 buffer solutions is shown in FIGS. 63A-D. Two16-solution multiplexer arrays, actuated by 8 control lines, allow forthe selection of buffers (left) and reagents (bottom). A PCI junction,formed by a 3-valve peristaltic injection pump and cross-injectionvalves dispenses directly into a 5 nL ring reactor.

Once the reactor has been flushed, a reagent line is selected and thecross-injection sequence is executed. The extended split channel regionincreases the volume of the cross-injection junction, thereby allowingfor up to 15 injections between flushing steps. The maximum number ofconsecutive injections that may be executed before the junction needs tobe refreshed depends on the Taylor dispersion of the injected fluid asit is pumped down the channel, and is therefore a function of theviscosity. The Taylor dispersion is discussed by Taylor, Proc. RoyalSoc. London Series a-Mathematical and Physical Sciences 219, 186-203(1953), incorporated by reference herein for all purposes,

FIG. 63B shows the injection of 4 slugs, each having a volume of 80 pL,into the ring reactor. Arbitrary combinations of 16 reagents may beproduced in the reactor by sequential flushing and injection steps.

FIG. 63C shows a color gradient formed from injections of water, bluedye, green dye, yellow dye, and red dye. In screening applications thatrequire the interrogation of a precious sample against many pre-mixedreagent formulations, the cross-injection flushing step is wasteful andis circumvented by the addition of a separate sample injection site, asshown in FIG. 63D.

After the ring is filled with the desired reagents, they are mixed byactuating a rotary peristaltic pump, as described by Chu et al.

The precision of metering was evaluated by injecting variable amounts ofdye (bromophenol blue sodium salt; Sigma) into a reactor, mixing, andperforming absorption measurements. FIG. 64A plots absorptionmeasurements showing high precision and reproducibility of PCIinjections. Each of the 9 clusters represents 100 identical injectionsequences.

The set of 900 sequential titration experiments shown in FIG. 64A showsthe metering to be both precise and reproducible, with a slope of 83.4pL per injection cycle and a coefficient of correlation of 0.996. Thestandard deviation of the injected slug volume was determined to beapproximately 0.6 pL. Although positive displacement metering ensuresthat the injected volume is robust to changes in the fluid viscosity,the viscosity of the working fluid does reduce the bandwidth of theinjector. It was found that for a solution having viscosity of 400 cPthe frequency response of the injector began to roll off at 10 Hz. Whenoperating at an injection frequency of 5 Hz all solutions havingviscosities below 400 cP produced equal injection volumes. Since themetering mechanism is completely mechanical, there is no dependence onthe pH or ionic strength of the injected fluid. Additionally, since thefluid is not dispensed from the chip, there is no phase interface, andtherefore little dependence on surface tension, so that the meteringtechnique is truly robust to the physical properties of the injectedfluid.

FIG. 64B plots absorption measurements of 4 sets of 20 injection andmixing sequences showing metering to be robust to the viscosity of theinjected fluid. Fluids contain varying amounts of glycerol and haveviscosity ranging from 1 cP to 400 cP. FIG. 64B indicates that titrationexperiments with fluids of varying glycerol concentration show theinjection volume to vary by less than 5% over a viscosity range of 1 cPto 400 cP without any modification to the injection sequence. Both FIGS.64A-B show precise and robust microfluidic metering.

In order to demonstrate the utility of ab initio solubilitycharacterization prior to crystallization trials, we explored thesolubility behavior of a commercially available crystallizationstandard, Endo-1,4-O-xylanase (xylanase) from Trichoderma reesei(Hampton Research). See Torronen et al., Embo Journal 13, 2493-2501(1994); and Torronen et al., Biochemistry 34, 847-856 (1995), both ofwhich are incorporated by reference herein for all purposes. Thestandard deviation of imaged pixels was used as a metric ofprecipitation, allowing for distinction between precipitated and solubleconditions and a rough quantitative measure of the degree ofprecipitation. Specifically, precipitation of the protein wasautomatically detected by imaging a portion of the mixing ring,calculating the standard deviation of the pixel intensities andcomparing this value to the background (no protein added). To ensureeven illumination, images were taken at 112 times magnification at a 9um high section of the mixing ring having rectangular cross-section.[0556] FIGS. 65A-C show automated exploration of protein solubilityusing microfluidic formulator. FIG. 65A shows precipitation measurementsat varying concentration of xylanase in 0.6 M Potassium Phosphate with0.1 M TRIS/HCl pH 6.5. Standard deviation of pixels provides aquantitative metric of protein precipitation. Below the precipitationlimit standard deviation shows constant background level with lowvariation. Above 12 mg/mL solution is in the precipitation regime wherethe pixel standard deviation exhibits an approximately linear dependenceon protein concentration. All points represent the mean of 5 identicalexperiments with error bars indicating standard deviation ofmeasurements. FIG. 65A shows that beyond the precipitation limit, thepixel standard deviation increases linearly with the proteinconcentration, and therefore is proportional to the concentration ofprecipitated protein present in the solution.

A two step protocol was used to map out the solubility space. An initialcoarse search identified reagents that have strong precipitating effectson the target macromolecule. This generates a solubility fingerprint ofthe crystallization target. Each precipitation peak in this fingerprintrepresents a chemical condition that exerts a pronounced effect onsolubility.

FIG. 65B shows solubility fingerprints of Xylanase over approximately4200 chemical conditions. The solubility fingerprint of Xylanase of FIG.65B was generated by 4 independent runs, each consisting ofapproximately 4000 titration experiments. Each data series represents aseparate fingerprinting experiment using the same basis of chemicalconditions. The crystallization conditions of FIG. 65B include thefollowing groups: combinations of salts as major precipitants at pHvalues from 4 to 9; and PEGS with salts at various pH values.

The top solubility fingerprint of FIG. 65B generated using a samplehaving elevated protein concentration (90 mg/mL), exhibits both highersignal to noise and additional peaks not present in the other dataseries (70 mg/mL). The two center solubility fingerprints were generatedsequentially on a single device (first the center top fingerprint, thenthe center bottom fingerprint) with the same loaded sample,demonstrating the stability of the protein over the time of theexperiment (approximately 20 hours). The bottom solubility fingerprintwas generated using the same sample as the top fingerprint but on aseparate device, showing reproducibility of the results across differentdevices.

Each solubility fingerprint was generated over a period of approximately35 hours and consumed approximately 8 μL of protein sample. Chemicalformulations were created by flushing the ring with one of 16 buffers,injecting a precipitating agent (salt or polymer), diluting the ringwith water, and then mixing. Protein sample was then introduced at avariety of concentrations and mixed prior to data acquisition. When apolymer was used as the major precipitating agent, a small amount ofsalt was also introduced as an additive (i.e. NaCl in FIGS. 65C22-23).

Experiments in FIG. 65B were grouped by the identity of the majorprecipitating agent so that each peak represents the effect of thisreagent over a range of pH values and concentrations. The large width ofthese peaks indicates robustness and a high level of experimentalredundancy, suggesting that a more efficient search could be conductedusing less related chemical conditions. Specifically, a large peak widthreveals that a large number of experiments with related conditionsyielded precipitation (i.e. all pH values resulted in precipitation whenthe precipitant is a phosphate salt). The search is thus inefficientsearch (too exhaustive), and the experiments would be more powerful ifmore sparse in nature (i.e. sampling other multi-component and unrelatedmixtures).

The solubility fingerprint is highly reproducible and is characteristicof the protein studied. For example, sodium chloride is a strongprecipitating agent (and effective crystallization agent) for anotherwell-studied crystallization standard (chicken egg white lysosyme) butdoes not produce a precipitation peak in the solubility fingerprint ofXylanase over the pH range studied. Thus with reference to theprecipitation curves shown in FIGS. 65C22-23 and discussed below, sodiumchloride is present as an additive only, with the major precipitantbeing PEG.

The solubility fingerprint of Xylanase revealed 5 salts (sodium citrate,di-potassium phosphate, ammonium sulfate, and sodium/potassium tartrate)as likely crystallizing agents. A high molecular weight polymer(polyethelyne glycol, M.W. 8,000) in combination with various saltadditives was also identified to be a strong precipitating agent at highpH values. The high isoelectric point of xylanase suggests that thereduced effectiveness of this precipitant at low pH values is duetwo-body electrostatic repulsion. A smaller molecular weight polymer(polyethelyne glycol, M.W. 3,350) was found to be a much weakerprecipitating agent and was not investigated further in phase-spacemapping experiments.

Chemical combinations identified as effective to yield precipitation inFIG. 65B were then employed to map a two dimensional phase space ofprotein/precipitant concentrations. Specifically, the identifiedXylanase precipitating conditions resulted in twenty-four expandedsystematic grid searches over all accessible protein and precipitantconcentrations. Each grid comprised seventy-two separate mixingexperiments, creating a two-dimensional phase-space with proteinconcentration and precipitant concentration as variables. Alltwenty-four phase spaces were generated sequentially on a single deviceusing less than 3 uL of protein sample (approximately 100 nL per phasespace) and are shown as FIGS. 65C1-24. In FIGS. 65C1-24, the size of thediamonds reflect the magnitude of standard deviation of the pictures,and hence provide a quasi-quantitative measurement of the amount ofprecipitation observed.

FIG. 65D shows comparison of phase mapping done on chip and inconventional microbatch experiments utilizing Na/K Tartrate as the firstprecipitant stock. This comparison of a precipitation phase spacesmeasured for Xylanase in chip (5 nL reactions) and in microbatch formatunder paraffin oil (5 μL reactions) shows good agreement in detectingthe precipitation boundary.

Since measurements of precipitation are made immediately after mixing(within 3 seconds), the locus of points that separate the precipitatedand soluble regions of the graph generate a precipitation curve that isdistinct from the thermodynamic solubility curve. Conditions that residejust below the precipitated region may be in a metastable stateconducive to crystallization.

A detailed knowledge of protein solubility behavior provides anempirical basis for the design of maximum likelihood crystallizationtrials. For example, the 24 phase spaces generated for Xylanase shown inFIGS. 65C1-24 were used to design an optimal crystallization screencomprising the 48 reagent combinations shown in TABLE 2.

TABLE 2 Precipitant Additive Xylanase Buffer (100 mM) Conc. Conc. Conc.Crystals Observed? FIG. # Cond. # pH Identity Identity (mM) Identity(mM) (mg/mL) Sample #1 Sample #2 65C15 1 4.6 NaCitrate NaCitrate 650 — 07 N N 65C15 2 4.6 NaCitrate NaCitrate 475 — 0 17 Y N 65C14 3 6.5Tris/HCl NaCitrate 700 — 0 5 Y N 65C14 4 6.5 Tris/HCl NaCitrate 500 — 09 Y N 65C13 5 8.45 Tris/HCl NaCitrate 425 — 0 19 N N 65C13 6 8.45Tris/HCl NaCitrate 475 — 0 9.5 Y N 65C2 7 4.6 NaCitrate Na/K Tartrate1100 — 0 6.5 N N 65C9 8 4.6 NaCitrate K₂HPO₄ 800 — 0 9 Y N 65C9 9 4.6NaCitrate K₂HPO₄ 1800 — 0 6.75 N N 65C10 10 6.5 Tris/HCl K₂HPO₄ 600 — 024.75 N N 65C1 11 6.5 Tris/HCl Na/K Tartrate 750 — 0 21 N N 65C11 128.45 Tris/HCl K₂HPO₄ 2800 — 0 6.75 N N 65C4 13 4.6 NaCitrate (NH₄)₂SO₄1080 — 0 9 N N 65C5 14 6.5 Tris/HCl (NH₄)₂SO₄ 1890 — 0 4.5 N N 65C6 158.45 Tris/HCl (NH₄)₂SO₄ 810 — 0 24.75 Y N 65C9 16 4.6 NaCitrate K₂HPO₄2800 — 0 3.5 N N 65C2 17 4.6 NaCitrate Na/K Tartrate 750 — 0 17 Y N65C11 18 8.45 Tris/HCl K₂HPO₄ 400 — 0 31.5 N N 65C4 19 4.6 NaCitrate(NH₄)₂SO₄ 945 — 0 23.625 N N 65C1 20 6.5 Tris/HCl Na/K Tartrate 1400 — 04.5 N N 65C5 21 6.5 Tris/HCl (NH₄)₂SO₄ 1350 — 0 9 Y N 65C3 22 8.45Tris/HCl Na/K Tartrate 900 — 0 6.75 Y N 65C3 23 8.45 Tris/HCl Na/KTartrate 700 — 0 13.5 Y Y 65C6 24 8.45 Tris/HCl (NH₄)₂SO₄ 1755 — 0 4.5 NN 65C22 25 8.2 Tris/HCl P8000 16000 NaCl 100 42 Y Y 65C22 26 8.2Tris/HCl P8000 23000 NaCl 100 21 Y Y 65C24 27 8.2 Tris/HCl P8000 15000 —0 54 Y Y 65C24 28 8.2 Tris/HCl P8000 24000 — 0 30 Y N 65C20 29 8.2Tris/HCl P8000 28000 NH₄CH₃CO₂ 100 18 Y N 65C20 30 8.2 Tris/HCl P800019000 NH₄CH₃CO₂ 100 33 Y Y 65C20 31 8.2 Tris/HCl P8000 14000 NH₄CH₃CO₂100 60 Y Y — 32 8.2 Tris/HCl P8000 20000 K Citrate 50 42 Y Y — 33 8.2Tris/HCl P8000 14000 K Citrate 50 60 Y Y — 34 8.2 Tris/HCl P8000 16000 KCitrate 50 36 Y Y 65C19 35 8.2 Tris/HCl P8000 24000 (NH₄)₂SO₄ 675 18 N Y65C7 36 8.2 Tris/HCl P8000 24000 (NH₄)₂SO₄ 675 6 N N 65C17 37 8.2Tris/HCl P8000 10000 MgSO₄ 50 66 Y Y 65C17 38 8.2 Tris/HCl P8000 16000MgSO₄ 50 30 Y Y 65C17 39 8.2 Tris/HCl P8000 18000 MgSO₄ 50 36 Y Y 65C2340 7.6 Tris/HCl P8000 12000 NaCl 100 66 Y Y 65C23 41 7.6 Tris/HCl P800028000 NaCl 100 18 N N 65C12 42 7.6 Tris/HCl P8000 30000 K₂HPO₄ 100 12 NY 65C12 43 7.6 Tris/HCl P8000 22000 K₂HPO₄ 100 12 N N 65C12 44 7.6Tris/HCl P8000 16000 K₂HPO₄ 100 42 Y Y 65C21 45 7.6 Tris/HCl P8000 18000NH₄CH₃CO₂ 100 36 Y Y 65C18 46 7.6 Tris/HCl P8000 28000 K Citrate 50 18 NY 65C8 47 7.6 Tris/HCl P8000 16000 (NH₄)₂SO₄ 675 54 N N 65C16 48 7.6Tris/HCl P8000 30000 MgSO₄ 50 12 Y N

Specifically, the empirically determined solubility boundaries of FIGS.65C1-24 were explored with crystallization trials, therebyeliminating 1) useless experiments on chemicals that do not altersolubility significantly (and hence will not produce crystals), and 2)useless experiments that are either too supersaturated and result onlyin protein aggregate, or are too undersaturated and result in theprotein remaining in solution.

A single batch crystallization trial using the optimal screen was set bycombining relative amounts of protein and precipitant stock so that thefinal condition was located on the boundary of the precipitation region.Specifically, protein was mixed with precipitant under oil at a ratiothat places the final concentrations of protein and precipitant on theboundary of the empirically determined solubility curves.

The efficiency of this screen was evaluated by comparison with standardcommercially available sparse matrix screens (Crystal Screen I andCrystal Screen II available from Hampton Research of Aliso Viejo,Calif., and Wizard I and Wizard II available from Emerald Biostructuresof Bainbridge Island, Wash.)

Two batch crystallization trials of the 48 unique conditions listed inTABLE 2 were prepared for each of the 4 sparse matrix screens for atotal of 384 individual assays. For each commercial screen final proteinconcentrations of 12.5 mg/mL and 25 mg/mL were used; the recommendedconcentration range for the crystallization of Xylanase is 10 mg/mL to40 mg/mL.

FIGS. 67-68 compare microbatch crystallization experiments usingcommercially available sparse matrix screens to an optimalcrystallization screen based on solubility phase spaces. FIG. 67 is ahistogram showing number of successful crystallization conditionsidentified with sparse matrix screens (each at protein concentrations of12 mg/mL and 23 mg/mL) and optimal screen.

Twenty-seven crystallization conditions were observed in the optimalscreen compared to a total of 3 crystallization conditions in the 8standard sparse matrix screens. The use of ab initio solubilityinformation therefore resulted in a 72-fold enrichment incrystallization success.

A surprising result was that Xylanase crystals were observed in theoptimal screen for all the precipitants identified in coarse screening.These results suggest that achieving optimal levels of supersaturationmay be more important in the crystallization of Xylanase than the broadsampling of chemical space. In cases such as this, systematic screeningfor crystallization using a reduced chemical space may prove moreeffective than sparse matrix strategies.

FIG. 68 is a polarized micrograph of large single crystals growndirectly from optimal screen (16% polyethelyne glycol 8000, 65 mM sodiumchloride, 65 mM TRIS-HCl pH 8.2, 42 mg/mL Xylanase). FIG. 68 shows thatcertain crystallization conditions identified in the optimal screen gavelarge single three-dimensional crystals, whereas only flat plateclusters were observed in the standard screens.

In order to evaluate the influence of lot variability on thesecrystallization results, the crystallization trials based on the optimalscreen of 48 reagent conditions, were repeated using new protein sampleobtained from the same vendor, and prepared in the same way as theoriginal sample. TABLE 2 also summarizes conditions under which Xylanasecrystals were observed to form from at least one of the two batches ofsample.

TABLE 2 indicates that certain crystallization conditions may be morerobust to batch-dependent perturbations. Specifically, as indicated withunderlining in TABLE 2, fourteen of seventeen polyethelyne glycol (PEG8000) conditions yielding crystals in the original experiment werereproduced using the second Xylanase sample. By contrast, only one often of the salt based conditions yielding crystals in the originalexperiment were reproduced using the second sample.

To determine if the highly variable crystallization behavior observed insalt-based conditions was due to variations in phase-space behavior acomplete phase-space of one chemical formulation (sodium/potassiumtartrate, TRIS.HCl pH 8.5) was measured in microbatch format for bothsamples. The plots of FIGS. 69A-B compare phase space behavior andcrystallization variability of the original and second samplesrespectively, in microbatch format. Conditions yielding no precipitateand no crystals are shown as circles. Conditions yielding crystal growthfrom clear solution are shown as diamonds. Conditions yielding crystalgrowth from immediate precipitate are shown as circles overlaid withdiamonds. Conditions yielding immediate precipitate with no crystalgrowth are shown as squares.

FIGS. 69A-B indicate that although both samples exhibited very similarphase-space behavior, they produced different crystallization results.Eleven conditions produced crystals in the original sample compared toonly one successful condition in the second sample. The reason for thisdifference in behavior is unclear but may be due to variable degrees ofproteolysis, or trace amounts of chemical contaminants introduced duringpurification or concentration steps.

Another application of protein solubility phase space mapping is intransporting successful crystallization conditions from one experimentalformat to another. The successful crystallization of a protein isdetermined both by the established thermodynamic variables and thekinetic trajectory of an experiment. For this reason experimentsconducted with different crystallization kinetics (eg. Hanging dropvapor diffusion, microbatch, free-interface diffusion) using the sameprecipitating agents will not necessarily produce similar results. Forexample, the hydroxylase domain of a cytochrome p450 alkane hydroxylase(Mutant 139-3 of BM-3) did not produce crystals in initial hanging droptrials, but was found to crystallize readily by microfluidic freeinterface diffusion (24) (1 part protein 20 mg/mL, 1 part 30% m/vpolyethelyne glycol 8000, 0.2 M sodium acetate, 0.1 M TRIS-HCl pH 7.0).This condition was, however, unsuccessful when set in hanging drop vapordiffusion format, resulting only in amorphous precipitate. Themicrofluidic formulator was used to generate a phase space at constantbuffer and salt concentration (100 mM TRIS-HCl pH 7.3; 200 mM sodiumacetate) with polyethelene glycol concentration and proteinconcentration as variables.

Two hanging drop experiments were designed to equilibrate near thesolubility limit determined from the phase space map. One condition (8μL of 35 mg/mL protein sample mixed with 6.7 μL of 10% polyetheleneglycol, 100 mM sodium acetate, 50 mM TRIS HCl pH 7.3, and equilibratedat 20° C. against 1 mM of 20% polyethelene glycol, 200 mm sodiumacetate, 100 mM TRIS-HCl pH 7.3) produced crystals within 3 days. Thissuccess demonstrates the usefulness of solubility mapping intransporting conditions across crystallization formats.

Finally, we also used the formulator to make a direct observation of thesupersaturation region of chicken egg white lysozyme. The concentrationsof salt and lysozyme was manipulated while keeping the bufferconcentration constant in order to evolve the chemical state of themixing ring radially out from the origin and then back again.Measurements of precipitation were taken at approximately 1 minuteintervals.

The addition of a family of such radial titrations was used to generatetwo phase space diagrams for chicken egg white lysozyme; one for theoutward titrations and one for the return titrations. FIG. 66 shows anoverlay of two phase-space diagrams generated by outward and returntitrations. Observed hysteresis in precipitation threshold identifiesmetastable region of phase space.

The first observation of protein precipitation appears at higher saltand protein concentration during the outward trajectory (increasingtarget material concentration) than on the return path (decreasingtarget material concentration), thereby exhibiting solubilityhysteresis. The intersection of the soluble region of the outward phasespace with the precipitated region of the return path phase spaceprovides a direct observation of a metastable regime in which theaggregate phase is thermodynamically stable but not observed at shorttimes. The observation of the reversible formation of a proteinaggregate may be used to distinguish between denatured and well-foldedprotein aggregates. Additionally, the identified metastable regions inphase space provide likely candidate conditions for crystal seeding andgrowth experiments.

In conclusion, we have shown that complex sample processing at thenanoliter scale allows for a practical implementation of automatedprotein solubility characterization. Ab initio solubility informationobtained through systematic protein phase space mapping provides aphysical basis for the design of optimal crystallization screens, givingrise to dramatic enrichment in crystallization success.

It must be noted that chemical conditions and phase behavior are not theonly variables that can be adjusted in the search for good crystals—itis often equally important to tune the properties of the protein bycreating mutants with terminal amino acids removed. However, the path tocrystallization always includes extensive chemical screening withprecious protein sample, and for this step it appears that microfluidicformulations devices can play an important role. Beyond applications inprotein crystallization the formulation capability of this device shouldfind diverse applications in areas such as combinatorial chemistry,chemical synthesis, and cell culture studies.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

1. An apparatus for investigating crystallization comprising: amicrofluidic formulator comprising a microfluidic chamber having arotary flow chamber configured to receive a crystallization target and aprecipitant; a light source configured to illuminate the microfluidicchamber; and a light detector configured to receive light transmittedthrough the microfluidic chamber.
 2. The apparatus of claim 1 furthercomprising a peristaltic pump actuable to flow the crystallizationtarget and crystallizing agent through the rotary flow chamber.
 3. Theapparatus of claim 1 wherein the detector comprises a plurality ofpixels configured to receive light transmitted through a portion of therotary flow chamber.
 4. The apparatus of claim 1 wherein the detectorcomprises a charge coupled device.
 5. The apparatus of claim 1 furthercomprising a processor configured to receive an electronic signal fromthe light detector indicating an intensity of light transmitted throughthe chamber and received on the pixel, the processor comprising acomputer readable storage medium having recorded instructions tocalculate from the electronic signal a standard deviation of pixelintensity of light transmitted through the portion, and to compare thestandard deviation to a background value recorded in an absence of thecrystallization target.
 6. An apparatus for investigatingcrystallization comprising: a chip holder for holding a microfluidicchamber having a rotary flow chamber configured to receive acrystallization target and a precipitant; a light source configured toilluminate the microfluidic chamber; and a light detector configured toreceive light transmitted through the microfluidic chamber.
 7. Theapparatus of claim 6 wherein the detector comprises a charge coupleddevice.
 8. The apparatus of claim 6 further comprising a processoradapted to receive an electronic signal from the light detectorindicating an intensity of light transmitted through a microfluidicchamber.